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hypopnea syndrome in rats
Respiratory Physiology & Neurobiology 136 (2003) 199 /209 www.elsevier.com/locate/resphysiol
Collapsible upper airway segment to study the obstructive sleep apnea/hypopnea syndrome in rats Ramon Farre´ a,*, Mar Rotger a, Josep M. Montserrat b, Gabriel Calero b, Daniel Navajas a a b
Unitat de Biofı´sica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Casanova 143, E-08036 Barcelona, Spain Institut Clı´nic de Pneumologia i Cirurgia Tora`cica, Institut d’Investigacions Biome`diques August Pi Sunyer, Barcelona, Spain Accepted 8 October 2002
Abstract Animal models have been used to study the pathophysiology of the obstructive sleep apnea/hypopnea syndrome (SAHS). Nevertheless, in none of the models described to date have the animals been subjected to the different patterns of upper airway obstructive events (apneas, hypopneas, and inspiratory flow limitation) characterizing SAHS. Our aim was to devise and test a computer-controlled collapsible upper airway segment applicable to rats and able to realistically mimic obstructive SAHS events. The collapsible segment (total volume B/2 cm3 and a dead space of :/0.25 cm3) consisted of a Starling resistor based on a latex membrane subjected to an external pressure applied by a computercontrolled pressure source. The collapsible segment was tested in eight anaesthetized and tracheostomized rats. The upper airway segment allowed us to induce obstructive apneas and hypopneas with flow and inspiratory effort waveforms similar to the ones observed in patients with SAHS. This collapsible upper airway segment may be a useful tool to implement a rat model of SAHS. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Disease, obstructive sleep apnea/hypopnea; Model, collapsible upper airway; Resistance, upper airways, Starling resistor; Upper airways, obstruction
1. Introduction The sleep apnea/hypopnea syndrome (SAHS) is characterized by recurrent airway obstructions resulting in breathing flow reductions, blood oxygen desaturations and arousals. Although the
* Corresponding author. Tel.: /34-93-402-4515; fax: /3493-402-4516. E-mail address: [email protected] (R. Farre´).
mechanisms involved in the pathogenesis of SAHS (Deegan and McNicholas, 1995) are not fully understood, there is ample evidence that an abnormal increase in upper airway collapsibility (Gold and Schwartz, 1996) is an important factor contributing to the development of the apneas, hypopneas and inspiratory flow limitation characterizing SAHS (Condos et al., 1994; Montserrat et al., 1995). Several animal models have been proposed to investigate both the pathophysiologic mechanisms
1569-9048/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1569-9048(03)00082-X
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involved in SAHS and the respiratory and cardiovascular consequences of the associated sleep disorders. Rodent models of chronic intermittent hypoxia induced by modifying the composition of the breathing gas have been used to investigate the effects of periodic oxygen desaturations (Fletcher et al., 1992; Bakehe et al., 1996; Bao et al., 1997; Fletcher et al., 1999; Greenberg et al., 1999; McGuire and Bradford, 1999; Kanagy et al., 2001; Tahawi et al., 2001). Other studies in sleeping (Issa et al., 1987; Hendricks et al., 1993; Kimoff et al., 1994; O’Donnell et al., 1994; Brooks et al., 1997; Schneider et al., 2000) or anesthetized (Scharf et al., 1992; White et al., 1995; O’Donnell et al., 1996a; Slamowitz et al., 1999; Chen and Scharf, 2000) larger animals have investigated the effects of recurrent obstructive apneas artificially provoked by closing the airway. However, to date no animal model has focused on the comparative study of the consequences of obstructive apneas, hypopneas and flow limitation events such as the ones observed in patients with SAHS. This lack of data is probably due to the absence of a procedure to induce these different kinds of obstructive events in animals. Given the fact that upper airway obstruction is the direct cause of the breathing disorders (increased breathing efforts, intermittent hypoxia, sleep architecture disruption) resulting in SAHS sequelae, animal models investigating the effects of obstructive events similar to those found in SAHS would be of great interest. Implementation of such a model in small animals would considerably facilitate research from the practical point of view. The aim of this work was to devise and
Fig. 1. Diagram of the upper airway collapsible segment. V? and Ptr indicate pressure ports to measure breathing flow and tracheal pressure, respectively. See text for explanation.
test a collapsible upper airway segment able to induce realistic obstructive events of controlled severity in rats. The collapsible segment devised was based on the principle of a Starling resistor. The degree of inspiratory obstruction was determined by application of computer-controlled pressure to the external side of a conduit with flexible walls. The implemented collapsible segment was assessed in the bench under realistic conditions and its applicability was tested in rats.
2. Methods 2.1. Description of the upper airway collapsible segment The collapsible upper airway segment devised to induce realistic obstructive events in rats (Fig. 1) consisted of two identical cylindrical chambers (1.8 mm in height and 13 mm diameter; internal dimensions) separated by a circular flexible membrane (132 mm2). The wall thickness of the cylindrical chambers was 1.5 mm. The membrane was cut from a conventional rubber latex examination glove (E300-M Safeskin-LPE, KimberlyClark, Zaventem, Belgium). The base of one of the chambers (left in Fig. 1) had a tube that was 9.5 mm long (l) with an internal diameter (ID) of 1.3 mm for connection to a source of external pressure (Pext). A tube (l/12 mm, ID /1.3 mm) at the center of the base of the other chamber (right in Fig. 1) allowed connection of the collapsible segment to the rat trachea. Another identical tube in this chamber wall was open to the atmosphere and acted as a pneumotachograph. The external diameter of these tubes was 2.5 mm. A pressure port built in this tube allowed the measurement of breathing flow (V?). A pressure port in the tube connecting the collapsible segment to the trachea (Fig. 1) was used to measure tracheal pressure (Ptr). The magnitude of the dynamic obstruction imposed by the collapsible segment was controlled by applying Pext to the collapsible segment by means of a computer-driven pressure source (Fig. 2). A small (8 /4 /4 cm3) domestic aquarium air pump (Nathura MK-701, 2.5 W; ECIS, Bressan-
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Fig. 2. Diagram of the computer-controlled system to apply an external pressure to the upper airway collapsible segment (see text for explanation).
vido, Italy) generated a pulsating flow with an amplitude which was controlled by means of a microcomputer and customized circuitry. The pump outlet was connected to the collapsible upper airway segment by means of a flexible tube (l /100 cm, ID /2 mm), allowing the pump to be placed at a convenient distance from the collapsible segment. The outlet of the pump was also connected to a tube (l /100 cm, ID /1 mm) that was open to the atmosphere (Fig. 2). This device allowed the application of any controlled Pext value between 0 and 80 cmH2O. Pressures at the ports of the collapsible segment (V? and Ptr in Fig. 1) were measured with piezoresistive transducers (PC176, Honeywell, Freeport, IL, USA) connected to the corresponding pressure ports (Fig. 1) by short and narrow tubes (l /25 cm, ID /1 mm). These signals were analogically low-pass filtered (Butterworth, eight poles, 8 Hz), sampled at 100 Hz and stored in the same microcomputer controlling the application of Pext to the collapsible segment (Fig. 2). 2.2. Characterization of the upper airway collapsible segment on the bench Estimation of actual flow V? from the pressure recorded at the corresponding port of the collapsible segment required prior characterization and calibration of the pressure /flow relationship in the
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tube acting as a pneumotachograph. To this end, an airflow source was used to apply different values of inspiratory and expiratory constant flow through the tracheal port of the collapsible segment (Pext /0). Actual flow was measured by means of a reference pneumotachograph (Fleisch000, Metabo, Epalinges, Switzerland) placed in series between the flow source and the tracheal port of the collapsible segment. The pressure drop in the pneumotachograph was measured with a differential transducer (Validyne MP-45, Northbrook, CA, USA). The relationship between flow and pressure at the port of the collapsible segment was characterized by fitting a parabolic model equation to data. The calibration equations corresponding to inspiration and expiration were subsequently used to compute V? from pressure recorded at the corresponding port in the collapsible segment. The mechanical properties of the upper airway collapsible segment were characterized in a bench test. A controlled negative pressure source connected to the tracheal port of the collapsible segment was used to impose different levels of constant negative Ptr. The relationship between Ptr and V? was assessed for values of Ptr ranging from 0 to /60 cmH2O and for values of Pext up to 50 cmH2O. The capability of the collapsible segment to induce static and dynamic obstructions similar to those found in patients with SAHS was also assessed in the bench. The tracheal port of the collapsible segment was connected to a system simulating the respiratory muscle pump of a rat. This system was based on a rodent mechanical ventilator (type 683, Harvard Apparatus, Marlborough, MA, USA). The room air inlet of the ventilator and its outlet to animal were both connected to the tracheal port of the collapsible segment. The ventilator was set to typical values of rat tidal volume (1 ml) and breathing frequency (100 breath/sec) (Strohl et al., 1997). V? and Ptr during the ventilator cycling were recorded for values of Pext ranging from 0 to 50 cmH2O. Given that the described collapsible segment was homemade, the variability in the performance of six different collapsible segments built in the lab was investigated.
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2.3. Application of the collapsible upper airway segment in rats The practical performance of the upper airway collapsible segment was assessed in eight Sprague/ Dawley rats weighing 250/300 g. The study was approved by the Animal Research Ethical Committee of the University of Barcelona. Each rat was anesthetized with an intraperitoneal injection of urethane (1.2 g/kg). A tracheotomy was performed and a cannula (2 mm ID) was inserted into the trachea. A pressure catheter was inserted into the carotid artery and arterial pressure (Pa) was recorded by means of a piezoresistive transducer (Uniflow, Baxter, Deerfield, IL, USA). The Pa signal was filtered and sampled as described for V? and Ptr. The tracheal port of the upper airway collapsible segment (Pext /0) was connected to the tracheal cannula of the rat. After 15 min of baseline breathing, the rat was subjected to a variety of controlled obstructive events induced by Pext patterns with different amplitude, duration and periodicity. At the end of the experiment, which lasted :/2 h, an abdominal aortic cannula was placed through a midline incision, heparin was administered, and the animal was sacrificed by exanguination.
3. Results 3.1. Characterization of the upper airway collapsible segment in the bench Fig. 3 shows the performance of a collapsible segment under constant inspiratory pressures for different Pext values. When Pext /0, the V?/Ptr relationship was virtually linear and the upper airway collapsible segment imposed a small resistive load to breathing (0.3 cmH2O sec/ml). As Pext was increased, the V?/Ptr relationship became more curvilinear. For values of Pext ]/10 cmH2O, a clear pattern of flow limitation was observed. Indeed, beyond a certain level of inspiratory pressure, flow remained constant regardless of reducing Ptr. The maximum inspiratory flow achieved inversely depended on the value of Pext. When Pext ]/50 cmH2O, inspiratory flow was
Fig. 3. Characterization of the upper airway collapsible segment under stationary conditions for different values of the external pressure (Pext) applied to the collapsible segment. V? is inspiratory flow and Ptr is tracheal pressure.
virtually nil for whole range of Ptr applied. The six different home-made collapsible segments showed a V?/Ptr relationship qualitatively similar to the one shown in Fig. 3. The only difference between the devices was in the value of the Pext required to obtain a given V?/Ptr curve. These differences in Pext, which were within 10 cmH2O, were easily compensated by setting the corresponding value in the computer-controlled system generating Pext (Fig. 2). Illustrative examples of the V? and Ptr signals obtained as the collapsible segment (same as in Fig. 3) was applied to a ventilator simulating a breathing rat are shown in Fig. 4. When Pext /0 the collapsible segment imposed a low load on breathing, as indicated by the small tracheal pressure swing required to generate the breathing flow. When Pext was increased to 25 cmH2O, a typical inspiratory flow limitation pattern appeared. At mid inspiration flow was almost constant regardless of the increasing negative tracheal pressure. During expiration the collapsible segment imposed a small load on breathing, as reflected by the low tracheal pressure required to induce the expiratory flow. As Pext was further increased to 35 cmH2O, the inspiratory load imposed on breathing was considerably increased,
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Fig. 4. Performance of the upper airway collapsible segment under dynamic conditions for different values of the external pressure (Pext) applied to the collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
which would correspond to a severe obstructive hypopnea. Finally, when Pext was increased to 50 cmH2O, a virtual obstructive apnea was induced. 3.2. Application of the upper airway collapsible segment in rats In agreement with the results obtained in the bench, application of the collapsible segment to all the rats allowed the induction of controlled upper airway obstructive events similar to the ones found in patients with SAHS. Fig. 5 shows an example illustrating that obstructive hypopneas of different magnitude were induced in a rat by setting the computer-controlled Pext applied to the collapsible segment. A Pext /30 cmH2O induced a mild hypopnea characterized by a 50% reduction in V?
with a marked increase in the Ptr swing. A more severe obstructive hypopnea (considerable reduction in V? and increase in Ptr swing) was achieved by setting Pext at 35 cmH2O. A virtual apnea (negligible flow despite the fact that the rat progressively increased the breathing effort) was induced by setting Pext at 40 cmH2O. The upper airway collapsible segment also allowed the induction of events with a time varying degree of obstruction, as the ones typically observed in patients with SAHS. Fig. 6 shows an example where the application of a time varying Pext induced an obstructive event with a time pattern characteristic of SAHS. The event started with a mild hypopnea, which increased in severity until virtual apnea and finished suddenly, resuming normal breathing. The pattern of the rat’s
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Fig. 6. Obstructive event of varying magnitude in an anesthetized rat. The time dependent severity of the obstruction was achieved by applying a varying external pressure (Pext) to the upper airway collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
Fig. 5. Obstructive events in an anesthetized rat. Hypopneas of different magnitude were induced by modifying the external pressure (Pext) applied to the upper airway collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
breathing effort during this event was also characteristic of SAHS: the pressure swing generated by the rat muscles progressively increased as the
degree of obstruction progressed, resuming normal values at the end of the obstructive event. Fig. 7 is an example illustrating that application of controlled upper airway obstructive events induced changes in the arterial blood pressure of the rat. The V? pattern in the figure shows that the animal was consecutively subjected to three hypopneas of different magnitude and to four apneas. Each obstructive event induced a transient change in arterial blood pressure. Pa increased during the event and recovered the baseline value after the end of the upper airway obstruction event. In this example the Pa swings induced by apneas and by severe hypopneas were of similar amplitude.
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Fig. 7. Changes in arterial pressure (Pa) in an anesthetized rat as a result of the application of a series of different obstructive events. V? is flow (V?/0 during inspiration).
4. Discussion To the best of our knowledge the collapsible upper airway segment devised in this work is the only system described to date for realistically mimicking the upper airway obstructive events characterizing patients with SAHS in animals. By selecting a suitable pattern of Pext, we were able to subject the rats to obstructions resulting in the same alterations of breathing flow, inspiratory muscle effort and arterial pressure observed in patients with SAHS. Indeed, rat ventilation (V?) and inspiratory effort (Ptr) during the obstructive events imposed by the collapsible segment (Figs. 5 and 6) exhibited the same waveform patterns as described in patients (Condos et al., 1994; Montserrat et al., 1995; Navajas et al., 1998). Moreover, the changes in arterial blood pressure (Fig. 7) also reproduced the transient alterations resulting from airway obstructions in animals (O’Donnell et al., 1996b; Chen and Scharf, 2000) and in patients with SAHS (Plane`s et al., 2002). The mechanics of upper airway obstruction in SAHS is commonly interpreted in terms of the Starling resistor model (Gold and Schwartz, 1996;
Farre´ et al., 1997, 1998). Accordingly, it is assumed that the upper airway is a conduit with a compliant wall and that the airway is open only when the transmural pressure across the wall is positive, i.e. when intraluminal pressure is greater than the external pressure. Inspiratory flow induces a negative intraluminal pressure owing to the resistance of the airway segment from the atmosphere to the collapsible section. Therefore, the degree of airway closure at any time depends on the balance between the negative intraluminal pressure and the pressure at the external side of the airway wall. In healthy subjects there is no inspiratory obstruction because the upper airway muscles exert sufficiently negative pressure on the external airway wall. Nevertheless, in patients with SAHS, the effective pressure applied by upper airway muscles is not sufficient to compensate the negative intraluminal pressure and, consequently, the airway is totally (apnea) or partially (hypopnea/flow limitation) occluded. The upper airway collapsible segment was devised to realistically mimic the mechanical properties of the upper airway in patients with SAHS. We ruled out the use of a fixed resistance
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such as an orifice (Greenberg et al., 1995; Gea et al., 2000) since this does not allow flow limitation and dynamic collapse. In order to achieve obstructive loads similar to the ones found in SAHS, we implemented a Starling resistor (Fig. 1). When no pressure was applied to the external side of the membrane (left chamber in Fig. 1), the membrane remained at its central position. As there was no airway obstruction, airflow could easily move from the atmosphere to the trachea (inspiration) and vice versa (expiration). However, when a positive Pext was applied to the collapsible segment, the flexible membrane was displaced towards the base of the chamber connected to the trachea. Therefore, the resistance of the conduit (right chamber in Fig. 1) tended to augment as Pext was increased. During expiration, the airflow entering the conduit from the trachea tended to restore the membrane to its central position with the result that air could easily be expired. By contrast, during inspiration, the negative intraluminal pressure owing to the upstream resistance tended to further displace the membrane away from its central position, thereby inducing partial or total obstruction at the entrance of the tube connected to the trachea. The degree of inspiratory obstruction was determined by the Pext applied. In addition to inducing upper airway obstruction, the collapsible segment allowed the easy monitoring of physiologic variables which were relevant to quantify the magnitude of the obstructive events experienced by the rat. Ptr was a direct measure of the extra effort required to breathe against the imposed load. V? was a measure of the breathing flow obtained from a small pneumotachograph (Fig. 1). When compared with conventional reference pneumotachographs, the one built in the collapsible segment had the advantages of simplicity and robustness and the drawbacks of nonlinearity and inspiration/expiration asymmetry. These drawbacks were overcome by linearizing the pressure signal (Farre´ et al., 2001) and by separately calibrating the inspiratory and expiratory flows. It should be pointed out, however, that to ensure adequate functioning of the collapsible segment and to avoid artifacts in estimating Ptr (Farre´ et al., 1989), the pressure measuring system should have a high impedance (transducer with a
low compliance sensing surface and tubing with reduced ID). The collapsible segment was able to induce realistic upper airway obstruction with a relatively low increase in the breathing dead space and in the baseline load to breathe. The dead space of the collapsible segment (right part in Fig. 1) was 0.25 ml. However, the effective dead space was lower taking into account the anatomic dead space corresponding to the by-passed upper airway of the rat. It should be noted that the dead space of the collapsible segment can be reduced by building a smaller collapsible segment, as we verified by constructing a prototype with reduced cylindrical chambers (1.5 mm in height and 9 mm diameter). When this smaller collapsible segment (dead space :/0.10 ml) was tested in the bench, it exhibited a behavior almost coincident with the one reported in Figs. 3 and 4. The only difference was that the Pext required were higher (up to 150 cmH2O) owing to the decrease in membrane compliance due to the area reduction. The use of this smaller collapsible segment in rats would only require increasing the power of the pump generating Pext (Fig. 2). The load to breathe imposed by the unobstructed collapsible segment at base line (Pext /0) was small. As shown in Fig. 3, achieving a typical inspiratory V?/5 ml/sec required a Ptr of less than 2 cmH2O, which corresponded to an added resistance slightly lower than the mechanical load of a rat respiratory system (Gomes et al., 2000). Given the fact that the baseline resistance of the collapsible segment is mainly due to their tubes (connection to trachea and pneumotachograph; Fig. 1), this resistance could be reduced by /50% by simply increasing the tubes ID from 1.3 to 1.6 mm, with a negligible increase in the dead space. The system employed to generate the Pext for regulating the degree of airway obstruction induced by the collapsible segment (Fig. 2) was based on the same rationale as in the conventional continuous positive airway pressure devices used to treat SAHS patients. Indeed, Pext was generated by the passage of an airflow through a resistor open to the atmosphere. The simple pump used generated an oscillating pressure at 50 Hz. Nevertheless, the pressure effectively applied to the collapsible segment was virtually constant given
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that the connecting tube plus the air inside the chamber of the collapsible segment and the membrane compliance behaved as a very efficient pneumatic low-pass filter (Farre´ et al., 2002). To facilitate modification of the pattern of Pext when designing and testing the system, Pext was controlled by a microcomputer (Fig. 2). However, for routine applications with a reduced number of different dynamic obstruction patterns, the microcomputer could be replaced by a simple circuit generating the desired signal patterns. The application of the devised collapsible segment in anesthetized rats will enable us to study the short term effects of realistic upper airway obstruction events, in particular, to investigate the changes in the production of the relevant signaling biomolecules observed in patients with SAHS (Noguchi et al., 1997; Ip et al., 2000; Ohga et al., 1999; Phillips et al., 1999). Comparison of the consequences of imposing realistic upper airway obstructions and of inducing intermittent hypoxia by modifying the breathing air (Fletcher, 2001) would be useful to improve our understanding of the specific role of mechanisms other than hypoxia in SAHS. Although anesthesia is a condition very different from sleep, short term experiments in anesthetized/sedated animals have provided relevant information on basic mechanisms of SAHS (Scharf et al., 1992; White et al., 1995; O’Donnell et al., 1996a; Slamowitz et al., 1999; Chen and Scharf, 2000). One advantage of inducing obstructive apneas/hypopneas during anesthesia is that comparison of the results obtained in sleeping and in anesthetized animals may provide some insight into the specific role played by arousals. For instance, this may be helpful to better understand the interaction between mechanical and neural mechanisms in inducing changes in arterial pressure in SAHS (Davis et al., 1993). Finally, it should be noted that after redesigning and testing, the collapsible segment could be applicable to a rat model with chronic respiratory sleep disturbances. This potential approach would allow us to more realistically study SAHS in rats since simultaneous implementation of EEG monitoring (Tagaito et al., 2001) would facilitate application/release of upper airway obstruction synchronized to sleep/ arousal.
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Acknowledgements The authors wish to thank Dr Ana Serrano (Consejo Superior de Investigaciones Cientı´ficas) and Miguel A. Rodrı´guez for their assistance. This work was supported in part by Ministerio Espan˜ol de Ciencia y Tecnologı´a (SAF2002-03616), Sociedad Espan˜ola de Neumologı´a y Cirugı´a Tora´cica (SEPAR-2001).
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Montserrat, J.M., Ballester, E., Olivi, H., Reolid, A., Lloberes, P., Morello, A., Rodriguez-Roisin, R., 1995. Time-course of stepwise CPAP titration: behavior of respiratory and neurological variables. Am. J. Respir. Crit. Care Med. 152, 1854 /1859. Navajas, D., Farre´, R., Rotger, M., Badia, R., Puig-deMorales, M., Montserrat, J.M., 1998. Assessment of airflow obstruction during CPAP by means of forced oscillation in patients with sleep apnea. Am. J. Respir. Crit. Care Med. 157, 1526 /1530. Noguchi, T., Chin, K., Ohi, M., Kita, H., Otsuka, N., Tsuboi, T., Satoh, M., Nakai, A., Kuno, K., Nagata, K., 1997. Heat shock protein 72 level decreases during sleep in patients with obstructive sleep apnea syndrome. Am. J. Respir. Crit. Care Med. 155, 1316 /1322. O’Donnell, C.P., King, E.D., Schwartz, A.R., Robotham, J.L., Smith, P.L., 1994. Relationship between blood pressure and airway obstruction during sleep in the dog. J. Appl. Physiol. 77, 1819 /1828. O’Donnell, C.P., Schwartz, A.R., Smith, P.L., Robotham, J.L., Fitzgerald, R.S., Shirahata, M., 1996a. Reflex stimulation of renal sympathetic nerve activity and blood pressure in response to apnea. Am. J. Respir. Crit. Care Med. 154, 1763 /1770. O’Donnell, C.P., Ayuse, T., King, E.D., Schwartz, A.R., Smith, P.L., Robotham, J.L., 1996b. Airway obstruction during sleep increases blood pressure without arousal. J. Appl. Physiol. 80, 773 /781. Ohga, E., Nagase, T., Tomita, T., Teramoto, S., Matsuse, T., Katayama, H., Ouchi, Y., 1999. Increased levels of circulating ICAM-1, VCAM-1, and L-selectin in obstructive sleep apnea syndrome. J. Appl. Physiol. 87, 10 /14. Phillips, B.G., Narkiewicz, K., Pesek, C.A., Haynes, W.G., Dyken, M.E., Somers, V.K., 1999. Effects of obstructive sleep apnea on endothelin-1 and blood pressure. J. Hypertens. 17, 61 /66. Plane`s, C., Leroy, M., Fayet, G., Aegerter, P., Foucher, A., Raffestin, A., 2002. Exacerbation of sleep-apnoea related nocturnal blood-pressure fluctuations in hypertensive subjects. Eur. Respir. J. 20, 151 /157. Scharf, S.M., Graver, L.M., Balaban, K., 1992. Cardiovascular effects of periodic occlusions of the upper airways in dogs. Am. Rev. Respir. Dis. 146, 321 /329. Schneider, H., Schaub, C.D., Chen, C.A., Andreoni, K.A., Schwartz, A.R., Smith, P.L., Robotham, J.L., O’Donnell, C.P., 2000. Neural and local effects of hypoxia on cardiovascular responses to obstructive apnea. J. Appl. Physiol. 88, 1093 /1102. Slamowitz, D., Chen, L., Scharf, S.M., 1999. Effects of vagotomy on cardiovascular response to periodic apneas in sedated pigs. J. Appl. Physiol. 86, 1890 /1896. Strohl, K.P., Thomas, A.J., St. Jean, P., Schlenker, E.H., Koletsky, R.J., Schork, N.J., 1997. Ventilation and metabolism among rat strains. J. Appl. Physiol. 82, 317 /323. Tagaito, Y., Polotsky, V.Y., Campen, M.J., Wilson, J.A., Balbir, A., Smith, P.L., Schwartz, A.R., O’Donnell, C.P.,
R. Farre´ et al. / Respiratory Physiology & Neurobiology 136 (2003) 199 /209 2001. A model of sleep-disordered breathing in the C57BL/ 6J mouse. J. Appl. Physiol. 91, 2758 /2766. Tahawi, Z., Orolinova, N., Joshua, I.G., Bader, M., Fletcher, E.C., 2001. Altered vascular reactivity in arterioles of
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chronic intermittent hypoxic rats. J. Appl. Physiol. 90, 2007 /2013. White, S.G., Fletcher, E.C., Miller, C.C., 1995. Acute systemic blood pressure elevation in obstructive and nonobstructive breath hold in primates. J. Appl. Physiol. 79, 324 /330.
Collapsible upper airway segment to study the obstructive sleep apnea/hypopnea syndrome in rats Ramon Farre´ a,*, Mar Rotger a, Josep M. Montserrat b, Gabriel Calero b, Daniel Navajas a a b
Unitat de Biofı´sica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona, Casanova 143, E-08036 Barcelona, Spain Institut Clı´nic de Pneumologia i Cirurgia Tora`cica, Institut d’Investigacions Biome`diques August Pi Sunyer, Barcelona, Spain Accepted 8 October 2002
Abstract Animal models have been used to study the pathophysiology of the obstructive sleep apnea/hypopnea syndrome (SAHS). Nevertheless, in none of the models described to date have the animals been subjected to the different patterns of upper airway obstructive events (apneas, hypopneas, and inspiratory flow limitation) characterizing SAHS. Our aim was to devise and test a computer-controlled collapsible upper airway segment applicable to rats and able to realistically mimic obstructive SAHS events. The collapsible segment (total volume B/2 cm3 and a dead space of :/0.25 cm3) consisted of a Starling resistor based on a latex membrane subjected to an external pressure applied by a computercontrolled pressure source. The collapsible segment was tested in eight anaesthetized and tracheostomized rats. The upper airway segment allowed us to induce obstructive apneas and hypopneas with flow and inspiratory effort waveforms similar to the ones observed in patients with SAHS. This collapsible upper airway segment may be a useful tool to implement a rat model of SAHS. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Disease, obstructive sleep apnea/hypopnea; Model, collapsible upper airway; Resistance, upper airways, Starling resistor; Upper airways, obstruction
1. Introduction The sleep apnea/hypopnea syndrome (SAHS) is characterized by recurrent airway obstructions resulting in breathing flow reductions, blood oxygen desaturations and arousals. Although the
* Corresponding author. Tel.: /34-93-402-4515; fax: /3493-402-4516. E-mail address: [email protected] (R. Farre´).
mechanisms involved in the pathogenesis of SAHS (Deegan and McNicholas, 1995) are not fully understood, there is ample evidence that an abnormal increase in upper airway collapsibility (Gold and Schwartz, 1996) is an important factor contributing to the development of the apneas, hypopneas and inspiratory flow limitation characterizing SAHS (Condos et al., 1994; Montserrat et al., 1995). Several animal models have been proposed to investigate both the pathophysiologic mechanisms
1569-9048/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1569-9048(03)00082-X
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involved in SAHS and the respiratory and cardiovascular consequences of the associated sleep disorders. Rodent models of chronic intermittent hypoxia induced by modifying the composition of the breathing gas have been used to investigate the effects of periodic oxygen desaturations (Fletcher et al., 1992; Bakehe et al., 1996; Bao et al., 1997; Fletcher et al., 1999; Greenberg et al., 1999; McGuire and Bradford, 1999; Kanagy et al., 2001; Tahawi et al., 2001). Other studies in sleeping (Issa et al., 1987; Hendricks et al., 1993; Kimoff et al., 1994; O’Donnell et al., 1994; Brooks et al., 1997; Schneider et al., 2000) or anesthetized (Scharf et al., 1992; White et al., 1995; O’Donnell et al., 1996a; Slamowitz et al., 1999; Chen and Scharf, 2000) larger animals have investigated the effects of recurrent obstructive apneas artificially provoked by closing the airway. However, to date no animal model has focused on the comparative study of the consequences of obstructive apneas, hypopneas and flow limitation events such as the ones observed in patients with SAHS. This lack of data is probably due to the absence of a procedure to induce these different kinds of obstructive events in animals. Given the fact that upper airway obstruction is the direct cause of the breathing disorders (increased breathing efforts, intermittent hypoxia, sleep architecture disruption) resulting in SAHS sequelae, animal models investigating the effects of obstructive events similar to those found in SAHS would be of great interest. Implementation of such a model in small animals would considerably facilitate research from the practical point of view. The aim of this work was to devise and
Fig. 1. Diagram of the upper airway collapsible segment. V? and Ptr indicate pressure ports to measure breathing flow and tracheal pressure, respectively. See text for explanation.
test a collapsible upper airway segment able to induce realistic obstructive events of controlled severity in rats. The collapsible segment devised was based on the principle of a Starling resistor. The degree of inspiratory obstruction was determined by application of computer-controlled pressure to the external side of a conduit with flexible walls. The implemented collapsible segment was assessed in the bench under realistic conditions and its applicability was tested in rats.
2. Methods 2.1. Description of the upper airway collapsible segment The collapsible upper airway segment devised to induce realistic obstructive events in rats (Fig. 1) consisted of two identical cylindrical chambers (1.8 mm in height and 13 mm diameter; internal dimensions) separated by a circular flexible membrane (132 mm2). The wall thickness of the cylindrical chambers was 1.5 mm. The membrane was cut from a conventional rubber latex examination glove (E300-M Safeskin-LPE, KimberlyClark, Zaventem, Belgium). The base of one of the chambers (left in Fig. 1) had a tube that was 9.5 mm long (l) with an internal diameter (ID) of 1.3 mm for connection to a source of external pressure (Pext). A tube (l/12 mm, ID /1.3 mm) at the center of the base of the other chamber (right in Fig. 1) allowed connection of the collapsible segment to the rat trachea. Another identical tube in this chamber wall was open to the atmosphere and acted as a pneumotachograph. The external diameter of these tubes was 2.5 mm. A pressure port built in this tube allowed the measurement of breathing flow (V?). A pressure port in the tube connecting the collapsible segment to the trachea (Fig. 1) was used to measure tracheal pressure (Ptr). The magnitude of the dynamic obstruction imposed by the collapsible segment was controlled by applying Pext to the collapsible segment by means of a computer-driven pressure source (Fig. 2). A small (8 /4 /4 cm3) domestic aquarium air pump (Nathura MK-701, 2.5 W; ECIS, Bressan-
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Fig. 2. Diagram of the computer-controlled system to apply an external pressure to the upper airway collapsible segment (see text for explanation).
vido, Italy) generated a pulsating flow with an amplitude which was controlled by means of a microcomputer and customized circuitry. The pump outlet was connected to the collapsible upper airway segment by means of a flexible tube (l /100 cm, ID /2 mm), allowing the pump to be placed at a convenient distance from the collapsible segment. The outlet of the pump was also connected to a tube (l /100 cm, ID /1 mm) that was open to the atmosphere (Fig. 2). This device allowed the application of any controlled Pext value between 0 and 80 cmH2O. Pressures at the ports of the collapsible segment (V? and Ptr in Fig. 1) were measured with piezoresistive transducers (PC176, Honeywell, Freeport, IL, USA) connected to the corresponding pressure ports (Fig. 1) by short and narrow tubes (l /25 cm, ID /1 mm). These signals were analogically low-pass filtered (Butterworth, eight poles, 8 Hz), sampled at 100 Hz and stored in the same microcomputer controlling the application of Pext to the collapsible segment (Fig. 2). 2.2. Characterization of the upper airway collapsible segment on the bench Estimation of actual flow V? from the pressure recorded at the corresponding port of the collapsible segment required prior characterization and calibration of the pressure /flow relationship in the
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tube acting as a pneumotachograph. To this end, an airflow source was used to apply different values of inspiratory and expiratory constant flow through the tracheal port of the collapsible segment (Pext /0). Actual flow was measured by means of a reference pneumotachograph (Fleisch000, Metabo, Epalinges, Switzerland) placed in series between the flow source and the tracheal port of the collapsible segment. The pressure drop in the pneumotachograph was measured with a differential transducer (Validyne MP-45, Northbrook, CA, USA). The relationship between flow and pressure at the port of the collapsible segment was characterized by fitting a parabolic model equation to data. The calibration equations corresponding to inspiration and expiration were subsequently used to compute V? from pressure recorded at the corresponding port in the collapsible segment. The mechanical properties of the upper airway collapsible segment were characterized in a bench test. A controlled negative pressure source connected to the tracheal port of the collapsible segment was used to impose different levels of constant negative Ptr. The relationship between Ptr and V? was assessed for values of Ptr ranging from 0 to /60 cmH2O and for values of Pext up to 50 cmH2O. The capability of the collapsible segment to induce static and dynamic obstructions similar to those found in patients with SAHS was also assessed in the bench. The tracheal port of the collapsible segment was connected to a system simulating the respiratory muscle pump of a rat. This system was based on a rodent mechanical ventilator (type 683, Harvard Apparatus, Marlborough, MA, USA). The room air inlet of the ventilator and its outlet to animal were both connected to the tracheal port of the collapsible segment. The ventilator was set to typical values of rat tidal volume (1 ml) and breathing frequency (100 breath/sec) (Strohl et al., 1997). V? and Ptr during the ventilator cycling were recorded for values of Pext ranging from 0 to 50 cmH2O. Given that the described collapsible segment was homemade, the variability in the performance of six different collapsible segments built in the lab was investigated.
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2.3. Application of the collapsible upper airway segment in rats The practical performance of the upper airway collapsible segment was assessed in eight Sprague/ Dawley rats weighing 250/300 g. The study was approved by the Animal Research Ethical Committee of the University of Barcelona. Each rat was anesthetized with an intraperitoneal injection of urethane (1.2 g/kg). A tracheotomy was performed and a cannula (2 mm ID) was inserted into the trachea. A pressure catheter was inserted into the carotid artery and arterial pressure (Pa) was recorded by means of a piezoresistive transducer (Uniflow, Baxter, Deerfield, IL, USA). The Pa signal was filtered and sampled as described for V? and Ptr. The tracheal port of the upper airway collapsible segment (Pext /0) was connected to the tracheal cannula of the rat. After 15 min of baseline breathing, the rat was subjected to a variety of controlled obstructive events induced by Pext patterns with different amplitude, duration and periodicity. At the end of the experiment, which lasted :/2 h, an abdominal aortic cannula was placed through a midline incision, heparin was administered, and the animal was sacrificed by exanguination.
3. Results 3.1. Characterization of the upper airway collapsible segment in the bench Fig. 3 shows the performance of a collapsible segment under constant inspiratory pressures for different Pext values. When Pext /0, the V?/Ptr relationship was virtually linear and the upper airway collapsible segment imposed a small resistive load to breathing (0.3 cmH2O sec/ml). As Pext was increased, the V?/Ptr relationship became more curvilinear. For values of Pext ]/10 cmH2O, a clear pattern of flow limitation was observed. Indeed, beyond a certain level of inspiratory pressure, flow remained constant regardless of reducing Ptr. The maximum inspiratory flow achieved inversely depended on the value of Pext. When Pext ]/50 cmH2O, inspiratory flow was
Fig. 3. Characterization of the upper airway collapsible segment under stationary conditions for different values of the external pressure (Pext) applied to the collapsible segment. V? is inspiratory flow and Ptr is tracheal pressure.
virtually nil for whole range of Ptr applied. The six different home-made collapsible segments showed a V?/Ptr relationship qualitatively similar to the one shown in Fig. 3. The only difference between the devices was in the value of the Pext required to obtain a given V?/Ptr curve. These differences in Pext, which were within 10 cmH2O, were easily compensated by setting the corresponding value in the computer-controlled system generating Pext (Fig. 2). Illustrative examples of the V? and Ptr signals obtained as the collapsible segment (same as in Fig. 3) was applied to a ventilator simulating a breathing rat are shown in Fig. 4. When Pext /0 the collapsible segment imposed a low load on breathing, as indicated by the small tracheal pressure swing required to generate the breathing flow. When Pext was increased to 25 cmH2O, a typical inspiratory flow limitation pattern appeared. At mid inspiration flow was almost constant regardless of the increasing negative tracheal pressure. During expiration the collapsible segment imposed a small load on breathing, as reflected by the low tracheal pressure required to induce the expiratory flow. As Pext was further increased to 35 cmH2O, the inspiratory load imposed on breathing was considerably increased,
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Fig. 4. Performance of the upper airway collapsible segment under dynamic conditions for different values of the external pressure (Pext) applied to the collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
which would correspond to a severe obstructive hypopnea. Finally, when Pext was increased to 50 cmH2O, a virtual obstructive apnea was induced. 3.2. Application of the upper airway collapsible segment in rats In agreement with the results obtained in the bench, application of the collapsible segment to all the rats allowed the induction of controlled upper airway obstructive events similar to the ones found in patients with SAHS. Fig. 5 shows an example illustrating that obstructive hypopneas of different magnitude were induced in a rat by setting the computer-controlled Pext applied to the collapsible segment. A Pext /30 cmH2O induced a mild hypopnea characterized by a 50% reduction in V?
with a marked increase in the Ptr swing. A more severe obstructive hypopnea (considerable reduction in V? and increase in Ptr swing) was achieved by setting Pext at 35 cmH2O. A virtual apnea (negligible flow despite the fact that the rat progressively increased the breathing effort) was induced by setting Pext at 40 cmH2O. The upper airway collapsible segment also allowed the induction of events with a time varying degree of obstruction, as the ones typically observed in patients with SAHS. Fig. 6 shows an example where the application of a time varying Pext induced an obstructive event with a time pattern characteristic of SAHS. The event started with a mild hypopnea, which increased in severity until virtual apnea and finished suddenly, resuming normal breathing. The pattern of the rat’s
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Fig. 6. Obstructive event of varying magnitude in an anesthetized rat. The time dependent severity of the obstruction was achieved by applying a varying external pressure (Pext) to the upper airway collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
Fig. 5. Obstructive events in an anesthetized rat. Hypopneas of different magnitude were induced by modifying the external pressure (Pext) applied to the upper airway collapsible segment. V? is flow (V?/0 during inspiration) and Ptr is tracheal pressure.
breathing effort during this event was also characteristic of SAHS: the pressure swing generated by the rat muscles progressively increased as the
degree of obstruction progressed, resuming normal values at the end of the obstructive event. Fig. 7 is an example illustrating that application of controlled upper airway obstructive events induced changes in the arterial blood pressure of the rat. The V? pattern in the figure shows that the animal was consecutively subjected to three hypopneas of different magnitude and to four apneas. Each obstructive event induced a transient change in arterial blood pressure. Pa increased during the event and recovered the baseline value after the end of the upper airway obstruction event. In this example the Pa swings induced by apneas and by severe hypopneas were of similar amplitude.
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Fig. 7. Changes in arterial pressure (Pa) in an anesthetized rat as a result of the application of a series of different obstructive events. V? is flow (V?/0 during inspiration).
4. Discussion To the best of our knowledge the collapsible upper airway segment devised in this work is the only system described to date for realistically mimicking the upper airway obstructive events characterizing patients with SAHS in animals. By selecting a suitable pattern of Pext, we were able to subject the rats to obstructions resulting in the same alterations of breathing flow, inspiratory muscle effort and arterial pressure observed in patients with SAHS. Indeed, rat ventilation (V?) and inspiratory effort (Ptr) during the obstructive events imposed by the collapsible segment (Figs. 5 and 6) exhibited the same waveform patterns as described in patients (Condos et al., 1994; Montserrat et al., 1995; Navajas et al., 1998). Moreover, the changes in arterial blood pressure (Fig. 7) also reproduced the transient alterations resulting from airway obstructions in animals (O’Donnell et al., 1996b; Chen and Scharf, 2000) and in patients with SAHS (Plane`s et al., 2002). The mechanics of upper airway obstruction in SAHS is commonly interpreted in terms of the Starling resistor model (Gold and Schwartz, 1996;
Farre´ et al., 1997, 1998). Accordingly, it is assumed that the upper airway is a conduit with a compliant wall and that the airway is open only when the transmural pressure across the wall is positive, i.e. when intraluminal pressure is greater than the external pressure. Inspiratory flow induces a negative intraluminal pressure owing to the resistance of the airway segment from the atmosphere to the collapsible section. Therefore, the degree of airway closure at any time depends on the balance between the negative intraluminal pressure and the pressure at the external side of the airway wall. In healthy subjects there is no inspiratory obstruction because the upper airway muscles exert sufficiently negative pressure on the external airway wall. Nevertheless, in patients with SAHS, the effective pressure applied by upper airway muscles is not sufficient to compensate the negative intraluminal pressure and, consequently, the airway is totally (apnea) or partially (hypopnea/flow limitation) occluded. The upper airway collapsible segment was devised to realistically mimic the mechanical properties of the upper airway in patients with SAHS. We ruled out the use of a fixed resistance
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such as an orifice (Greenberg et al., 1995; Gea et al., 2000) since this does not allow flow limitation and dynamic collapse. In order to achieve obstructive loads similar to the ones found in SAHS, we implemented a Starling resistor (Fig. 1). When no pressure was applied to the external side of the membrane (left chamber in Fig. 1), the membrane remained at its central position. As there was no airway obstruction, airflow could easily move from the atmosphere to the trachea (inspiration) and vice versa (expiration). However, when a positive Pext was applied to the collapsible segment, the flexible membrane was displaced towards the base of the chamber connected to the trachea. Therefore, the resistance of the conduit (right chamber in Fig. 1) tended to augment as Pext was increased. During expiration, the airflow entering the conduit from the trachea tended to restore the membrane to its central position with the result that air could easily be expired. By contrast, during inspiration, the negative intraluminal pressure owing to the upstream resistance tended to further displace the membrane away from its central position, thereby inducing partial or total obstruction at the entrance of the tube connected to the trachea. The degree of inspiratory obstruction was determined by the Pext applied. In addition to inducing upper airway obstruction, the collapsible segment allowed the easy monitoring of physiologic variables which were relevant to quantify the magnitude of the obstructive events experienced by the rat. Ptr was a direct measure of the extra effort required to breathe against the imposed load. V? was a measure of the breathing flow obtained from a small pneumotachograph (Fig. 1). When compared with conventional reference pneumotachographs, the one built in the collapsible segment had the advantages of simplicity and robustness and the drawbacks of nonlinearity and inspiration/expiration asymmetry. These drawbacks were overcome by linearizing the pressure signal (Farre´ et al., 2001) and by separately calibrating the inspiratory and expiratory flows. It should be pointed out, however, that to ensure adequate functioning of the collapsible segment and to avoid artifacts in estimating Ptr (Farre´ et al., 1989), the pressure measuring system should have a high impedance (transducer with a
low compliance sensing surface and tubing with reduced ID). The collapsible segment was able to induce realistic upper airway obstruction with a relatively low increase in the breathing dead space and in the baseline load to breathe. The dead space of the collapsible segment (right part in Fig. 1) was 0.25 ml. However, the effective dead space was lower taking into account the anatomic dead space corresponding to the by-passed upper airway of the rat. It should be noted that the dead space of the collapsible segment can be reduced by building a smaller collapsible segment, as we verified by constructing a prototype with reduced cylindrical chambers (1.5 mm in height and 9 mm diameter). When this smaller collapsible segment (dead space :/0.10 ml) was tested in the bench, it exhibited a behavior almost coincident with the one reported in Figs. 3 and 4. The only difference was that the Pext required were higher (up to 150 cmH2O) owing to the decrease in membrane compliance due to the area reduction. The use of this smaller collapsible segment in rats would only require increasing the power of the pump generating Pext (Fig. 2). The load to breathe imposed by the unobstructed collapsible segment at base line (Pext /0) was small. As shown in Fig. 3, achieving a typical inspiratory V?/5 ml/sec required a Ptr of less than 2 cmH2O, which corresponded to an added resistance slightly lower than the mechanical load of a rat respiratory system (Gomes et al., 2000). Given the fact that the baseline resistance of the collapsible segment is mainly due to their tubes (connection to trachea and pneumotachograph; Fig. 1), this resistance could be reduced by /50% by simply increasing the tubes ID from 1.3 to 1.6 mm, with a negligible increase in the dead space. The system employed to generate the Pext for regulating the degree of airway obstruction induced by the collapsible segment (Fig. 2) was based on the same rationale as in the conventional continuous positive airway pressure devices used to treat SAHS patients. Indeed, Pext was generated by the passage of an airflow through a resistor open to the atmosphere. The simple pump used generated an oscillating pressure at 50 Hz. Nevertheless, the pressure effectively applied to the collapsible segment was virtually constant given
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that the connecting tube plus the air inside the chamber of the collapsible segment and the membrane compliance behaved as a very efficient pneumatic low-pass filter (Farre´ et al., 2002). To facilitate modification of the pattern of Pext when designing and testing the system, Pext was controlled by a microcomputer (Fig. 2). However, for routine applications with a reduced number of different dynamic obstruction patterns, the microcomputer could be replaced by a simple circuit generating the desired signal patterns. The application of the devised collapsible segment in anesthetized rats will enable us to study the short term effects of realistic upper airway obstruction events, in particular, to investigate the changes in the production of the relevant signaling biomolecules observed in patients with SAHS (Noguchi et al., 1997; Ip et al., 2000; Ohga et al., 1999; Phillips et al., 1999). Comparison of the consequences of imposing realistic upper airway obstructions and of inducing intermittent hypoxia by modifying the breathing air (Fletcher, 2001) would be useful to improve our understanding of the specific role of mechanisms other than hypoxia in SAHS. Although anesthesia is a condition very different from sleep, short term experiments in anesthetized/sedated animals have provided relevant information on basic mechanisms of SAHS (Scharf et al., 1992; White et al., 1995; O’Donnell et al., 1996a; Slamowitz et al., 1999; Chen and Scharf, 2000). One advantage of inducing obstructive apneas/hypopneas during anesthesia is that comparison of the results obtained in sleeping and in anesthetized animals may provide some insight into the specific role played by arousals. For instance, this may be helpful to better understand the interaction between mechanical and neural mechanisms in inducing changes in arterial pressure in SAHS (Davis et al., 1993). Finally, it should be noted that after redesigning and testing, the collapsible segment could be applicable to a rat model with chronic respiratory sleep disturbances. This potential approach would allow us to more realistically study SAHS in rats since simultaneous implementation of EEG monitoring (Tagaito et al., 2001) would facilitate application/release of upper airway obstruction synchronized to sleep/ arousal.
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Acknowledgements The authors wish to thank Dr Ana Serrano (Consejo Superior de Investigaciones Cientı´ficas) and Miguel A. Rodrı´guez for their assistance. This work was supported in part by Ministerio Espan˜ol de Ciencia y Tecnologı´a (SAF2002-03616), Sociedad Espan˜ola de Neumologı´a y Cirugı´a Tora´cica (SEPAR-2001).
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