Researchin VeterinaryScience1991,51, 55-60
Mechanical properties of the isolated equine trachea T. ART, P. LEKEUX, Laboratory for Functional Investigation, Faculty of Veterinary Medicine, University of Libge, BMiment B42, Sart-Tilman, B-4000 Libge, Belgium
In order to study the in vitro mechanical properties of the equine trachea submitted to the compressive pressures observed in vivo, the pressure-volume relationship was determined in intra- and extrathoracic tracheal segments taken post mortem from 29 healthy horses (one to 15 years old; 352 to 651 kg). At the same time, the cross-sectional lumen area (XSA) at the mid-point of the segment was measured using a slit-lamp transillumination and photographic measurement by endoscopy. The tracheal specific compliance (Cs) as well as the relative changes in X-SA and in the sagittal and transverse diameters, for intraluminal pressures from 5 to - 5 kPa, were calculated. The extrathoracic tracheal Cs was 0.060 -4- 0.002 kPa-1 and, at an intraluminal pressure of - 5 kPa, XSA was reduced to about 73 per cent of its resting value. The intrathoracic segments were more compliant and, at similar compressive pressure, their X-SA was more reduced. These data show that the equine tracheal compliance is high and suggest that the increase in pulmonary resistance observed during strenuous exercise may he partly explained by a partial tracheal collapse.
IN a previous study, the authors studied the partitioning of the total pulmonary resistance in exercising ponies and reported that the increase of total pulmonary resistance during exertion was mainly due to the increase of the extrathoracic and intrathoracic airways resistance during inspiration and expiration, respectively (Art et al 1988). These observations led to the suggestion that, during exercise, a partial collapse of these airways may occur either during inspiration, for the extrathoracic airways, or during expiration, for the intrathoracic airways. Indeed, the tracheal lumen is determined at any given instance by its inherent compliance, ie, the ease with which it can be deformed, and the transmural pressure (TMP),that is, the difference between the intra-airway and the Reprint requests to Dr T. Art, Servicede Physiologie,Facultdde Mddecine Vdtdrinaire, Universitd de Li6ge, Batiment B42, Sart Tilman B-4000, Li6ge,Belgium 55
external pressures as a direct result of the deforming force exerted upon it (Wittenborg et al 1967). The TMP may be either compressive, when the intraluminal pressure is lower than the external one, or dilating in the other case. Compressive TMP reduces the intraluminal cross-sectional area (X-SA) and therefore induces an increase in airway resistance: this phenomenon occurs at the level of the extrathoracic airways during forced inspiration, in exercising horses or in patients suffering from upper airways obstruction, and at the level of the intrathoracic airways during forced expiration (Gillespie 1974). In human medicine, it is already well known that the dynamic behaviour of the extra- and intrathoracic airways is an important factor in modulating respiratory airflow. Indeed, it was previously reported that during forced expiration and coughing as well as during forced inspiration, the intra- and extrathoracic airways are submitted to high compressive transmural pressure and can narrow considerably (Dekker and Ledeboer 1961, Hoffstein et al 1980, Griscom and Wohl 1983, Brown et al 1986). Even during tidal respiration in normal subjects the alterations in the calibre of the airways have been shown to be partly related to the fluctuations in transmural gradients (Martin and Proctor 1958). In a previous study negative tracheal pressures of - 3 " 5 kPa were recorded in ponies performing moderate exercise, and more recently minimal pleural pressures of about - 5 kPa were observed in thoroughbred horses during strenuous exercise (Art et al 1990). These experimental data suggest that even in the healthy horse the airways are probably submitted to high compressive TMP during exercise and consequently flow rates could be limited by the dynamic compression inducing a partial collapse of the large airways. However, this hypothesis i s based on the supposition that the trachea is sufficiently compliant. Although the role of the compliance of the small airways in the increase of airflow resistance during forced expiration is already well known in large animals (Gillespie 1974), there is a lack of information concerning the mechanical properties of the large
56
T. Art, P. Lekeux
airways in the equine species and their ability to witlastand these high TMP. This fact led the authors to study the in vitro compliance of extrathoracic isolated tracheal segments submitted to the compressive pressures observed in vivo. The mechanical properties of intra- and extrathoracic segments were also compared.
Materials and methods
Preparation and maintenance of the airways Twenty-nine tracheal segments were removed from freshly slaughtered horses (one to 15 years old; weighing 352 to 651 kg). The horses were considered healthy on clinical and post mortem examinations. The segments were dissected and separated distal to the larynx and proximal to the tracheal bifurcation. They were immediately submerged in a cool (4 to 10°C) Ringer's lactate solution. The length of the trachea in situ and under longitudinal tension has been shown to be significantly greater than the length of the isolated trachea under no tension (Miserocchi and Agostoni 1973). Therefore, before removal, the length of each segment still in situ was measured with the neck of the horse in a normal position. Each segment was composed of 25 to 30 rings, that is from the first to the 25th/30th ring for the extrathoracic segments and from the 27th to the 52nd ring for the intrathoracic ones. The tracheas were studied 12 to 16 hours after the horses were killed. A preliminary study performed on five segments had shown that there was variability in the results (three repeated measurements obtained from each trachea) up to the 12th hour after death (coefficient of variation [cv] between 17.2 and 29-8). This phenomenon was probably due to variability in the state of contraction of the smooth tracheal muscle. Measurement of the adenosine triphosphate (ATe) content in frozen samples of smooth muscle taken from three segments indicated that after 12 hours tracheal smooth muscle was depleted in ATP and was no longer able to contract. Therefore, in order to standardise the state of contraction of the muscle and to minimise the variability due to any variations in the post mortem changes, experiments were conducted 12 hours after death. The influence of the variability of the tracheal smooth tone in fresh trachea has already been reported by Moreno et al (1986) and by Souhrada and Dickey (1976) who reported spontaneous contractions in isolated in vitro guinea pig trachea for a period of 10 to 12 hours after removing the segment from the animal. Short lengths of rigid PVC tube of the largest possible diameter (3 to 6 cm, according to the tracheal size) were inserted and securely tied to each end of the segment. These tubes were plugged with cylindrical
rubber corks. One plug was solid, the other had a central hole for passing a fibroscope through (Coloscope; Fuji), which protruded into the lumen in a standardised position (3 cm from the rubber cork). The fibroscope permitted visualisation of the airway lumen and was used to take photographs of the X-SA of the airway. Another hole in the same plug connected the lumen of the segment to the pressure and volume recording apparatus (Fig 1). During the experiment, the airway was tied securely to a rack for its normal length in order to prevent any lengthwise movement and, except during the mounting of the apparatus, it was wrapped in cotton-wool soaked in Ringer's lactate solution at room temperature.
Apparatus Studies were made with experimental apparatus (Fig 1) consisting of a 250 ml syringe, a differential pressure transducer (Statham, Gould), rigid connection to the airway and a polygraph (Gould ES 1000). Two slide projectors positioned and aligned on
4
t.~6
.[--1 FIG 1 : Apparatus for observing transilluminated tracheal lumen while simultaneously measuring the intraluminal pressure changes for any gradual changes in tracheal volume. 1 Tracheal segment, 2 Rubber cork, 3 Slide projectors, 4 Pressure transducer, 5 250 ml syringe, 6 Fibroscope, 7 Camera, 8 Polygraph
Mechanical properties of equine trachea opposite sides were used to direct a slit of light approximately 2 mm wide on to the same point of the trachea. When viewed through the fibroscope in a darkened room the circumference of the airway was outlined by transillumination. This apparatus allowed the changes in X-SA and geometry of the segment during the experiment to be photographed (Fujinon, Fuji; Tri-X pan, 400, Kodak). Photographs were taken at the level~of the 12th or 13th ring and of the 42nd or 43rd ring for the extrathoracic and intrathoracic segments, respectively.
57
were recorded. The changes in X-SA (measured by planimetry) as well as in the sagittal (os) and transverse (DT) diameters were measured on the photographs (Fig 2).
Experimental procedure
Before each study, the apparatus was checked for leaks and the absolute volume of the airways was measured. Afterwards, the tracheal segment was inflated at 2 kPa TMP for five minutes. The ability of the tracheal segment to hold a state of distension for this period confirmed the airtightness of the whole Measurements experimental fitting. Furthermore this manoeuvre Before the experiment, the absolute volume of the standardised the pre-treatment of each segment. airway lumen was measured with the fitting in place. A quasi-static pressure/volume measurement startThe absolute volume of the trachea was the average ing with TMP----0 was made with equal decrements or (five measurements) volume of Ringer's solution increments of volume (I0 ml) every 60 seconds. The which was withdrawn from the airways filled to its TMP was continuously recorded at a paper speed of resting length. This volume was measured while the 1 m m s - ~on the polygraph and an 'endophotograph' trachea was submerged in saline. of the X-SAwas taken after each step. A preliminary During the experiment, changes in TMP and in X-SA study assessed that the pressure changes related to induced by gradual changes of the syringe volume stress relaxation continued for about 30 minutes after the step gradient, but 92 per cent of this change occurred within 60 seconds. Thus changes were recorded after 60 seconds to take this into account. OS Gradual deflation of the airways up to TMP < -- 5 kPa was followed by gradual inflation, return to the 'resting' volume (Vo), gradual inflation up to TMP> + 5 kPa and then deflation to the Vo. A pressure of 5 kPa was chosen because this was considered as the physiological limit encountered in healthy running horses (Art et al 1990). In order to check the reproducibility of the tracheal response, the same protocol was repeated three times for each segment, the photographs being taken the third time. The coefficient of variation (cv) of the three sets of results was calculated and the results were discarded when cv was more than 5 per cent. The experiments were always carried out in the same sequence. First, 29 extrathoracic segments were investigated for their mechanical properties. Secondly, the same experimental procedure was used to compare the behaviour of extra- and intrathoracic segments (n = 6), each pair coming from the same horse.
~~1¢,0T
Calculations
FiG 2: Cross-sectional shape of one extrathoracic segment at a transmural pressure of 0 kPa (A), - 2" 51 kPa (B) and - 5- 4 9 kPa (C), respectively. DT and DS represent the transverse and sagittal diameters, respectively. Redrawn from the original photographs
Volume changes, expressed as the percentage of Vo, and the corresponding TMP were determined and plotted to provide individual quasi-static pressurevolume relationships (Fig 3). Specific airways compliance (Cs) value was determined as the relative change in Vo over a pressure range of 0 to - 5 kPa TMP on the deflation limb of the pressure/volume curve. This represents the linear portion of the deflation curve for all segments.
T. Art, P. Lekeux
58
TABLE 1 : Relative changes (given in per cent) in volume, transverse diameter (DT). sagittal diameter (DS) and DI'/DS ratio in extrathoracic tracheal segments at various transmural pressures (TMP) (mean ± SEM; n = 29 and 18 for the
compressive and dilating TMP, respectively) Dilating TMP 3 kPa Volume DT DS DT/DS
+11 .5 +0"05 +10"3 -10"6
± ± ± ±
Compressive TMP , - 3 kPa - 5 kPa
5 kPa 1.5 0"01 3"6 1 "5
+ 2 0 . 1 -~ 2 - 2 +0'02±0-01 +14.9 ± 2"6 - 1 1 "6 ± 2 " 3
The changes in X-SA, DS, DT and the DT/DS ratio induced by the gradual changes in the syringe volume were derived from the photographs taken by transillumination, and were divided by their corresponding values at TMP----0. The relative changes were related to changes in TMP and plotted on a graph. The relative changes so obtained in X-SA,DS, DT and DT/DS at various TMP were interpolated from these curves. Data are given as mean + standard error. Significance levels were assessed by an ANOVA test and probabilities < 0.05 were considered as significant. Results The mean extrathoracic tracheal Cs ( n = 29) was 0-060 -4-.0.002 kPa -~. At an intraluminal pressure of - 5 kPa, ×-SA was reduced to 73.2 =~ 1 "6 per cent of its resting value. This change in X-SAwas due to a decrease of DS (67"2 4- 2"8 per cent of its resting value) rather than to a decrease of DT (92"2 4- 10"0 per cent), as indicated in Table 1. Consequently, the DT/DS ratio was increased by a factor of 1.45 -4- 0.09. The relative changes in volume, DT, DS and DT/DS of the extrathoracic segments at various TMP are given in Table 1. These changes were more marked with compressive than with equivalent dilating TMP. Eleven of the 29 extrathoracic and five of the six intrathoracic segments were unable to sustain positive intraluminal pressures and presented emphysema in the sheath of connective tissue that lies externally. The comparison of the mechanical behaviour of six extrathoracic and their corresponding intrathoracic segments is shown in Table 2. Cs was significantly higher in the intrathoracic than in the extrathoracic
-19.2 ± 0.9 -4"4±0"7 +22"3 ± 1"9 +25 ± 3'8
-29.9 ± 0-9 -7"8±1-1 -32"8 ± 2"8 +44"8 ± 8"9
segments. The X-SA of the intrathoracic segments decreased more than X-SA of the extrathoracic segments and this reduction was due to a decrease in both DT and DS. Discussion Although the excised tracheal preparation does not perhaps mimic the in vivo behaviour of the trachea, it has been frequently used to provide valuable information on the dimensional, static and dynamic properties of the tracheal wall (Martin and Proctor 1958, Croteau and Cook 1961, Bobbaers et al 1978, Bhutani et al 1981). Some bronchoscopic observations of the intrathoracic tracheal lumen performed in vivo in anaesthetised cats (Tandler et al 1983) and in humans (Dekker and Ledeboer 1961) during forced expiration have suggested that supporting tissues which surround the trachea do not prevent the mechanism of collapse in vivo. The resistance to airflow of a cylindrical tube is inversely proportional to its radius to the fourth power. In this study, the X-SA of extrathoracic tracheal segments, subjected to a compressive TMP of 5 kPa was reduced to 73.2 per cent of their resting value. From a theoretical point of view, this means that when TMP 5 kPa their radii would be reduced to 85 per cent of their resting value and, therefore, that their resistance to airflow would be multiplied by 1.91. It must be pointed out that in a recent study performed on galloping thoroughbred horses, total pulmonary resistance has been found to be multiplied by about 2-7 (Art et al 1990). Although it is known that other factors contribute to the increase in total =
- -
TABLE 2: Comparison of the mechanical behaviour of six extrathoracic segments (ET) with their corresponding intrathoracic segments (IT). The changes in cross-sectional area (X-SA), in sagittal diameter (DS), in transverse diameter (DT) and in the DT/DS ratio at a compressive transmural pressure o f 5 kPa are expressed as percentage o f the value of each parameter at zero transmural pressure Cs ( k P a - 1 ) ET IT
0-O61 ~- 0 " 0 0 3 0"085*
± 0"007
X-SA (%) 74.3±1-8 59"5* ± 5"2
* Significantly different from ET values with P < O" 05. Cs Specific compliance. Mean ± SEM. n = 6
DT (%) 93"0±2'1 8 4 " 3 * 4- 1 "7
DS (%)
DT/DS (%)
73"5±4"2
128.5±9"3
68"3 ± 6"7
130-7 ± 15'2
Mechanical properties of equine trachea
10.
-10
-20
-30
-4O
- 7 -6
-5
- 4 -3 - 2 -1 0 kPa
1
2
3
4
5
6
FIG 3: Typical pressure/volume (vol) changes relationship from an extrathoracic segment. The diagram shows points of reference used to measure the volume changes given in Table 2. Arrows indicate the increasing and decreasing pressures. The curve, starting with transmural pressure (TMP) = 0 was obtained by gradual deflation of the airways up to TMP< - 5 kPa (D1), followed by a gradual inflation (11), return to the resting volume (Vo), gradual inflation (I2) up to TMP> 5 kPa and gradual deflation to the V 0 (D2). The discontinuity between the starting and ending points at V 0 is due to hysteresis
pulmonary resistance with exercise, ie, turbulence, inhomogeneous distribution of resistance among airways, etc, it may be suggested that, during tidal ventilation, a partial tracheal collapse, that is, the reduction of X-SA at the level of the extra- and intrathoracic trachea during inspiration and expiration, respectively, could partly explain the substantial
59
increase in pulmonary resistance. The photographs obtained by endoscopy in the present study showed clearly that the tracheal rings, which are incomplete posteriorly, allowed the collapse of the trachea by invagination of the posterior wall. Thus the soft tissue membrane contributes substantially to the pressure/ volume characteristics of the large airways. This was particularly true as regards extrathoracic segments where the 8 per cent reduction of DT and the 33 per cent reduction of DS at TMP = - - 5 kPa assesses the rigidity of the tracheal cartilage rings and the tendency of the tracheal muscle to protrude into the tracheal lumen. The collapse of the tracheal soft tissue has been shown to be reduced by hormone- or neurally-induced contraction of the tracheal smooth muscle (Olsen et al 1967, Coburn et al 1972, Knudson and Knudson 1975): although the force of contraction of the trachealis muscle is primarily circumferential, this contraction decreases the diameter of the trachea and forms a cartilaginous tube with a decreased compressibility. Knudson and Knudson (1975) and Bouhuys and Van de Woestijne (1971) have demonstrated that tracheal or bronchial muscle contraction can result in decreased flow conductance at low flows but increased flow rates at higher driving pressures when the less compliant trachea resists flow-limiting collapse. Because the hormonal response to exercise is mainly sympathetic, ie, increased plasma epinephrine and norepinephrine (Thornton 1985), it certainly does not induce any tracheal smooth muscle contraction. Moreover, previous studies have demonstrated that maximal or normal inflation of the lungs can temporarily abolish or reduce airway smooth muscle tone (Nadel and Tierney 1961, Drazen et al 1979, Gunst and Lai-Fook 1983). Pride et al (1967) and Knudson and Knudson (1975) pointed out that, once the airways collapse begins, the Bernoulli effect (Knudson and Knudson 1975) would begin to contribute, to a progressively increasing degree, to further diminution in airway X-SA. The changes in X-SA observed in the present work were simultaneous with dramatic changes in its shape: as there was no overlapping of the cartilage ends, compressive TMP results in a concave-convex X-SA (Fig 2). If the same is true in vivo, when the trachea is submitted to a compressive TMP, this change of shape might induce an increase in resistance by increasing frictional resistance and by promoting turbulence. The authors concluded that the large extmthoracic and intrathoracic airways are sufficiently compliant to be collapsible when submitted to high, but nevertheless physiological, compressive TMP and therefore that this in vitro study confirms the hypothesis previously suggested that the increase in total pulmonary resistance recorded in horses during strenuous exercise may be due to a partial collapse of the large airways.
T. A r t , P. L e k e u x
60 Acknowledgements
The authors wish to thank M. Delacroix for her helpful technical assistance and M. Leblond for typing the manuscript. We also thank Professor G. Maghuin-Rogister and G. Degand for their valuable advice and help with measuring the ATP content. References ART, T., SERTEYN, D. & LEKEUX, P. (1988) Effect of exercise on the partitioning of equine respiratory resistance. Equine Veterinary Journal 20, 268-273 ART, T., ANDERSON, L. S., ROBERTS, C. A., WOAKES, A. J., BUTLER, P. J., SNOW, D. H. & LEKEUX, P. (1990) Mechanics of breathing during strenuous exercise in thoroughbred horses. Respiration Physiology 82, 279-284 BHUTANI, V. K., RUBENSTEIN, S. D. & SHAFPER, T. H. (1981) Pressure-volume relationships of trachea in fetal newborn and adult rabbits. Respiration Physiology 41,221-231 BOBBAERS, H., CLEMENT, J. & VAN DE WOESTIJNE, K. P. (1978) Dynamic yiscoelastic properties of the canine trachea.
Journal of Applied Physiology: Respiration Environment and Exercise Physiology 44, 137-143 BOUHUYS, A. & VAN DE WOESTIJNE, K. P. (1971) Mechanical consequences of airway smooth muscle relaxation. Journal of Applied Physiology 30, 670-676 BROWN, I. G., WEBSTER, P. M., ZAMEL, N. & HOFFSTEIN, V. (1986) Changes in tracheal cross-sectional area during MueUer and Valsalva maneuvers in humans. Journal of Applied Physiology 60, 1865-1870 COBURN, R. F., THORNTON, D. & ARTS, R. (1972) Effect of trachealis muscle contraction on tracheal resistance to airflow. Journal of Applied Physiology 32, 397-403 CROTEAU, J. R. & COOK, C. D. (1961) Volume-pressure and length-tension measurements in human tracheal and bronchial segments. Journal of Applied Physiology 16, 170-172 DEKKER, E. & LEDEBOER, R. (1961) Compression of the tracheobronchial tree by the action of the voluntary respiratory musculature in normal individuals a n d in patients with asthma and emphysema. American Journal of Roentgenology, Radium Therapy and Nuclear Medicine 85, 217-228 DRAZEN, J. M., LORING, S. H., JACKSON, A. C., SNAPPER, J. R. & INGRAM, R. H. (1979) Effects of volume history on airway changes induced by histamine or vagal stimulation. Journal
GRISCOM, N. T. & WOHL, M. E. B. (1983) Tracheal size and shape: effects of change in intraluminal pressure. Radiology 149, 27-30 GUNST, S. J. & LAI-FOOK, S. J. (1983) Effect of inflation on trachealis muscle tone in canine tracheal segments in vitro. Journal
of Applied Physiology: Respiration, Environment and Exercise Physiology 54, 906-913 HOFFSTEIN, V., GLASS, G. M., WOHL, M. E., DORKIN, H. L., STRIEDER, D. J. & FREDBERG, J. J. (1980) Tracheal geometry and compliance during breathing. Physiologist 23, 165 KNUDSON, R. J. & KNUDSON, D. E. (1975) Effect of muscle constriction on flow-limiting collapse of isolated canine trachea. Journal of Applied Physiology 38, 125-131 MARTIN, B. H. & PROCTOR, D. F. (1958) Pressure-volume measurements on dog bronchi. Journal of Applied Physiology 13, 337-343 MISEROCCHI, G. & AGOSTONI, E. (1973) Longitudinal forces acting on the trachea. Respiration Physiology 17, 62-71 MORENO, R. H., McCORMACK, G. S., BRENDAN, J., MULLEN, M., HOGG, J. C., BERT, J. & PARE, P. D. (1986) Effect of intravenous papain on tracheal pressure-volume curves in rabbits. Journal of Applied Physiology 60, 247-252 NADEL, J. A. & TIERNEY, E. (1961) Effect of a previous deep inspiration on airway resistance in man. Journal of Applied Physiology 16, 717-719 OLSEN, C. R., STEVENS, A. E. & MclLROY, M. B. (1967) Rigidity of trachea and bronchi during muscular constriction. Journal of Applied Physiology 23, 27- 34 PRIDE, N. B., PERMUTT, S., RILEY, R. L. & BROMBERGERBARNEA, B. (1967) Determinants of maximum expiratory flow from the lungs. Journal of Applied Physiology 23, 646-662 SOUHRADA, J. F. & DICKEY, D. W. (1976) Mechanical activities of trachea as measured in vitro and in vivo. Respiration Physiology 26, 27-40 TANDLER, B., SHERMAN, J. M., BOAT, T. F. & WOOD, R. E. (1983) Surface architecture of the mucosal epithelium of the cat trachea: II. Structure and dynamics of the membranous portion. American Journal of Anatomy 168, 133-144 THORNTON, J. R. (1985) Hormonal responses to exercise and training. VeterinaryClinics of North America, Equine Practice 1, 477-498 WITTENBORG, M. H., GYEPES, M. T. & CROCKER, D. (1967) Tracheal dynamics in infants with respiratory distress, stridor, and collapsing trachea. Radiology 88, 653-662
of Applied Physiology: Respiration Environment and Exercise Physiology 47, 657-665 GILLESPIE, J. R. (1974) The role of the respiratory system during exertion. Journal of the South African VeterinaryAssociation 45, 305-309
Received July 11, 1990 Accepted January 21, 1991
Mechanical properties of the isolated equine trachea T. ART, P. LEKEUX, Laboratory for Functional Investigation, Faculty of Veterinary Medicine, University of Libge, BMiment B42, Sart-Tilman, B-4000 Libge, Belgium
In order to study the in vitro mechanical properties of the equine trachea submitted to the compressive pressures observed in vivo, the pressure-volume relationship was determined in intra- and extrathoracic tracheal segments taken post mortem from 29 healthy horses (one to 15 years old; 352 to 651 kg). At the same time, the cross-sectional lumen area (XSA) at the mid-point of the segment was measured using a slit-lamp transillumination and photographic measurement by endoscopy. The tracheal specific compliance (Cs) as well as the relative changes in X-SA and in the sagittal and transverse diameters, for intraluminal pressures from 5 to - 5 kPa, were calculated. The extrathoracic tracheal Cs was 0.060 -4- 0.002 kPa-1 and, at an intraluminal pressure of - 5 kPa, XSA was reduced to about 73 per cent of its resting value. The intrathoracic segments were more compliant and, at similar compressive pressure, their X-SA was more reduced. These data show that the equine tracheal compliance is high and suggest that the increase in pulmonary resistance observed during strenuous exercise may he partly explained by a partial tracheal collapse.
IN a previous study, the authors studied the partitioning of the total pulmonary resistance in exercising ponies and reported that the increase of total pulmonary resistance during exertion was mainly due to the increase of the extrathoracic and intrathoracic airways resistance during inspiration and expiration, respectively (Art et al 1988). These observations led to the suggestion that, during exercise, a partial collapse of these airways may occur either during inspiration, for the extrathoracic airways, or during expiration, for the intrathoracic airways. Indeed, the tracheal lumen is determined at any given instance by its inherent compliance, ie, the ease with which it can be deformed, and the transmural pressure (TMP),that is, the difference between the intra-airway and the Reprint requests to Dr T. Art, Servicede Physiologie,Facultdde Mddecine Vdtdrinaire, Universitd de Li6ge, Batiment B42, Sart Tilman B-4000, Li6ge,Belgium 55
external pressures as a direct result of the deforming force exerted upon it (Wittenborg et al 1967). The TMP may be either compressive, when the intraluminal pressure is lower than the external one, or dilating in the other case. Compressive TMP reduces the intraluminal cross-sectional area (X-SA) and therefore induces an increase in airway resistance: this phenomenon occurs at the level of the extrathoracic airways during forced inspiration, in exercising horses or in patients suffering from upper airways obstruction, and at the level of the intrathoracic airways during forced expiration (Gillespie 1974). In human medicine, it is already well known that the dynamic behaviour of the extra- and intrathoracic airways is an important factor in modulating respiratory airflow. Indeed, it was previously reported that during forced expiration and coughing as well as during forced inspiration, the intra- and extrathoracic airways are submitted to high compressive transmural pressure and can narrow considerably (Dekker and Ledeboer 1961, Hoffstein et al 1980, Griscom and Wohl 1983, Brown et al 1986). Even during tidal respiration in normal subjects the alterations in the calibre of the airways have been shown to be partly related to the fluctuations in transmural gradients (Martin and Proctor 1958). In a previous study negative tracheal pressures of - 3 " 5 kPa were recorded in ponies performing moderate exercise, and more recently minimal pleural pressures of about - 5 kPa were observed in thoroughbred horses during strenuous exercise (Art et al 1990). These experimental data suggest that even in the healthy horse the airways are probably submitted to high compressive TMP during exercise and consequently flow rates could be limited by the dynamic compression inducing a partial collapse of the large airways. However, this hypothesis i s based on the supposition that the trachea is sufficiently compliant. Although the role of the compliance of the small airways in the increase of airflow resistance during forced expiration is already well known in large animals (Gillespie 1974), there is a lack of information concerning the mechanical properties of the large
56
T. Art, P. Lekeux
airways in the equine species and their ability to witlastand these high TMP. This fact led the authors to study the in vitro compliance of extrathoracic isolated tracheal segments submitted to the compressive pressures observed in vivo. The mechanical properties of intra- and extrathoracic segments were also compared.
Materials and methods
Preparation and maintenance of the airways Twenty-nine tracheal segments were removed from freshly slaughtered horses (one to 15 years old; weighing 352 to 651 kg). The horses were considered healthy on clinical and post mortem examinations. The segments were dissected and separated distal to the larynx and proximal to the tracheal bifurcation. They were immediately submerged in a cool (4 to 10°C) Ringer's lactate solution. The length of the trachea in situ and under longitudinal tension has been shown to be significantly greater than the length of the isolated trachea under no tension (Miserocchi and Agostoni 1973). Therefore, before removal, the length of each segment still in situ was measured with the neck of the horse in a normal position. Each segment was composed of 25 to 30 rings, that is from the first to the 25th/30th ring for the extrathoracic segments and from the 27th to the 52nd ring for the intrathoracic ones. The tracheas were studied 12 to 16 hours after the horses were killed. A preliminary study performed on five segments had shown that there was variability in the results (three repeated measurements obtained from each trachea) up to the 12th hour after death (coefficient of variation [cv] between 17.2 and 29-8). This phenomenon was probably due to variability in the state of contraction of the smooth tracheal muscle. Measurement of the adenosine triphosphate (ATe) content in frozen samples of smooth muscle taken from three segments indicated that after 12 hours tracheal smooth muscle was depleted in ATP and was no longer able to contract. Therefore, in order to standardise the state of contraction of the muscle and to minimise the variability due to any variations in the post mortem changes, experiments were conducted 12 hours after death. The influence of the variability of the tracheal smooth tone in fresh trachea has already been reported by Moreno et al (1986) and by Souhrada and Dickey (1976) who reported spontaneous contractions in isolated in vitro guinea pig trachea for a period of 10 to 12 hours after removing the segment from the animal. Short lengths of rigid PVC tube of the largest possible diameter (3 to 6 cm, according to the tracheal size) were inserted and securely tied to each end of the segment. These tubes were plugged with cylindrical
rubber corks. One plug was solid, the other had a central hole for passing a fibroscope through (Coloscope; Fuji), which protruded into the lumen in a standardised position (3 cm from the rubber cork). The fibroscope permitted visualisation of the airway lumen and was used to take photographs of the X-SA of the airway. Another hole in the same plug connected the lumen of the segment to the pressure and volume recording apparatus (Fig 1). During the experiment, the airway was tied securely to a rack for its normal length in order to prevent any lengthwise movement and, except during the mounting of the apparatus, it was wrapped in cotton-wool soaked in Ringer's lactate solution at room temperature.
Apparatus Studies were made with experimental apparatus (Fig 1) consisting of a 250 ml syringe, a differential pressure transducer (Statham, Gould), rigid connection to the airway and a polygraph (Gould ES 1000). Two slide projectors positioned and aligned on
4
t.~6
.[--1 FIG 1 : Apparatus for observing transilluminated tracheal lumen while simultaneously measuring the intraluminal pressure changes for any gradual changes in tracheal volume. 1 Tracheal segment, 2 Rubber cork, 3 Slide projectors, 4 Pressure transducer, 5 250 ml syringe, 6 Fibroscope, 7 Camera, 8 Polygraph
Mechanical properties of equine trachea opposite sides were used to direct a slit of light approximately 2 mm wide on to the same point of the trachea. When viewed through the fibroscope in a darkened room the circumference of the airway was outlined by transillumination. This apparatus allowed the changes in X-SA and geometry of the segment during the experiment to be photographed (Fujinon, Fuji; Tri-X pan, 400, Kodak). Photographs were taken at the level~of the 12th or 13th ring and of the 42nd or 43rd ring for the extrathoracic and intrathoracic segments, respectively.
57
were recorded. The changes in X-SA (measured by planimetry) as well as in the sagittal (os) and transverse (DT) diameters were measured on the photographs (Fig 2).
Experimental procedure
Before each study, the apparatus was checked for leaks and the absolute volume of the airways was measured. Afterwards, the tracheal segment was inflated at 2 kPa TMP for five minutes. The ability of the tracheal segment to hold a state of distension for this period confirmed the airtightness of the whole Measurements experimental fitting. Furthermore this manoeuvre Before the experiment, the absolute volume of the standardised the pre-treatment of each segment. airway lumen was measured with the fitting in place. A quasi-static pressure/volume measurement startThe absolute volume of the trachea was the average ing with TMP----0 was made with equal decrements or (five measurements) volume of Ringer's solution increments of volume (I0 ml) every 60 seconds. The which was withdrawn from the airways filled to its TMP was continuously recorded at a paper speed of resting length. This volume was measured while the 1 m m s - ~on the polygraph and an 'endophotograph' trachea was submerged in saline. of the X-SAwas taken after each step. A preliminary During the experiment, changes in TMP and in X-SA study assessed that the pressure changes related to induced by gradual changes of the syringe volume stress relaxation continued for about 30 minutes after the step gradient, but 92 per cent of this change occurred within 60 seconds. Thus changes were recorded after 60 seconds to take this into account. OS Gradual deflation of the airways up to TMP < -- 5 kPa was followed by gradual inflation, return to the 'resting' volume (Vo), gradual inflation up to TMP> + 5 kPa and then deflation to the Vo. A pressure of 5 kPa was chosen because this was considered as the physiological limit encountered in healthy running horses (Art et al 1990). In order to check the reproducibility of the tracheal response, the same protocol was repeated three times for each segment, the photographs being taken the third time. The coefficient of variation (cv) of the three sets of results was calculated and the results were discarded when cv was more than 5 per cent. The experiments were always carried out in the same sequence. First, 29 extrathoracic segments were investigated for their mechanical properties. Secondly, the same experimental procedure was used to compare the behaviour of extra- and intrathoracic segments (n = 6), each pair coming from the same horse.
~~1¢,0T
Calculations
FiG 2: Cross-sectional shape of one extrathoracic segment at a transmural pressure of 0 kPa (A), - 2" 51 kPa (B) and - 5- 4 9 kPa (C), respectively. DT and DS represent the transverse and sagittal diameters, respectively. Redrawn from the original photographs
Volume changes, expressed as the percentage of Vo, and the corresponding TMP were determined and plotted to provide individual quasi-static pressurevolume relationships (Fig 3). Specific airways compliance (Cs) value was determined as the relative change in Vo over a pressure range of 0 to - 5 kPa TMP on the deflation limb of the pressure/volume curve. This represents the linear portion of the deflation curve for all segments.
T. Art, P. Lekeux
58
TABLE 1 : Relative changes (given in per cent) in volume, transverse diameter (DT). sagittal diameter (DS) and DI'/DS ratio in extrathoracic tracheal segments at various transmural pressures (TMP) (mean ± SEM; n = 29 and 18 for the
compressive and dilating TMP, respectively) Dilating TMP 3 kPa Volume DT DS DT/DS
+11 .5 +0"05 +10"3 -10"6
± ± ± ±
Compressive TMP , - 3 kPa - 5 kPa
5 kPa 1.5 0"01 3"6 1 "5
+ 2 0 . 1 -~ 2 - 2 +0'02±0-01 +14.9 ± 2"6 - 1 1 "6 ± 2 " 3
The changes in X-SA, DS, DT and the DT/DS ratio induced by the gradual changes in the syringe volume were derived from the photographs taken by transillumination, and were divided by their corresponding values at TMP----0. The relative changes were related to changes in TMP and plotted on a graph. The relative changes so obtained in X-SA,DS, DT and DT/DS at various TMP were interpolated from these curves. Data are given as mean + standard error. Significance levels were assessed by an ANOVA test and probabilities < 0.05 were considered as significant. Results The mean extrathoracic tracheal Cs ( n = 29) was 0-060 -4-.0.002 kPa -~. At an intraluminal pressure of - 5 kPa, ×-SA was reduced to 73.2 =~ 1 "6 per cent of its resting value. This change in X-SAwas due to a decrease of DS (67"2 4- 2"8 per cent of its resting value) rather than to a decrease of DT (92"2 4- 10"0 per cent), as indicated in Table 1. Consequently, the DT/DS ratio was increased by a factor of 1.45 -4- 0.09. The relative changes in volume, DT, DS and DT/DS of the extrathoracic segments at various TMP are given in Table 1. These changes were more marked with compressive than with equivalent dilating TMP. Eleven of the 29 extrathoracic and five of the six intrathoracic segments were unable to sustain positive intraluminal pressures and presented emphysema in the sheath of connective tissue that lies externally. The comparison of the mechanical behaviour of six extrathoracic and their corresponding intrathoracic segments is shown in Table 2. Cs was significantly higher in the intrathoracic than in the extrathoracic
-19.2 ± 0.9 -4"4±0"7 +22"3 ± 1"9 +25 ± 3'8
-29.9 ± 0-9 -7"8±1-1 -32"8 ± 2"8 +44"8 ± 8"9
segments. The X-SA of the intrathoracic segments decreased more than X-SA of the extrathoracic segments and this reduction was due to a decrease in both DT and DS. Discussion Although the excised tracheal preparation does not perhaps mimic the in vivo behaviour of the trachea, it has been frequently used to provide valuable information on the dimensional, static and dynamic properties of the tracheal wall (Martin and Proctor 1958, Croteau and Cook 1961, Bobbaers et al 1978, Bhutani et al 1981). Some bronchoscopic observations of the intrathoracic tracheal lumen performed in vivo in anaesthetised cats (Tandler et al 1983) and in humans (Dekker and Ledeboer 1961) during forced expiration have suggested that supporting tissues which surround the trachea do not prevent the mechanism of collapse in vivo. The resistance to airflow of a cylindrical tube is inversely proportional to its radius to the fourth power. In this study, the X-SA of extrathoracic tracheal segments, subjected to a compressive TMP of 5 kPa was reduced to 73.2 per cent of their resting value. From a theoretical point of view, this means that when TMP 5 kPa their radii would be reduced to 85 per cent of their resting value and, therefore, that their resistance to airflow would be multiplied by 1.91. It must be pointed out that in a recent study performed on galloping thoroughbred horses, total pulmonary resistance has been found to be multiplied by about 2-7 (Art et al 1990). Although it is known that other factors contribute to the increase in total =
- -
TABLE 2: Comparison of the mechanical behaviour of six extrathoracic segments (ET) with their corresponding intrathoracic segments (IT). The changes in cross-sectional area (X-SA), in sagittal diameter (DS), in transverse diameter (DT) and in the DT/DS ratio at a compressive transmural pressure o f 5 kPa are expressed as percentage o f the value of each parameter at zero transmural pressure Cs ( k P a - 1 ) ET IT
0-O61 ~- 0 " 0 0 3 0"085*
± 0"007
X-SA (%) 74.3±1-8 59"5* ± 5"2
* Significantly different from ET values with P < O" 05. Cs Specific compliance. Mean ± SEM. n = 6
DT (%) 93"0±2'1 8 4 " 3 * 4- 1 "7
DS (%)
DT/DS (%)
73"5±4"2
128.5±9"3
68"3 ± 6"7
130-7 ± 15'2
Mechanical properties of equine trachea
10.
-10
-20
-30
-4O
- 7 -6
-5
- 4 -3 - 2 -1 0 kPa
1
2
3
4
5
6
FIG 3: Typical pressure/volume (vol) changes relationship from an extrathoracic segment. The diagram shows points of reference used to measure the volume changes given in Table 2. Arrows indicate the increasing and decreasing pressures. The curve, starting with transmural pressure (TMP) = 0 was obtained by gradual deflation of the airways up to TMP< - 5 kPa (D1), followed by a gradual inflation (11), return to the resting volume (Vo), gradual inflation (I2) up to TMP> 5 kPa and gradual deflation to the V 0 (D2). The discontinuity between the starting and ending points at V 0 is due to hysteresis
pulmonary resistance with exercise, ie, turbulence, inhomogeneous distribution of resistance among airways, etc, it may be suggested that, during tidal ventilation, a partial tracheal collapse, that is, the reduction of X-SA at the level of the extra- and intrathoracic trachea during inspiration and expiration, respectively, could partly explain the substantial
59
increase in pulmonary resistance. The photographs obtained by endoscopy in the present study showed clearly that the tracheal rings, which are incomplete posteriorly, allowed the collapse of the trachea by invagination of the posterior wall. Thus the soft tissue membrane contributes substantially to the pressure/ volume characteristics of the large airways. This was particularly true as regards extrathoracic segments where the 8 per cent reduction of DT and the 33 per cent reduction of DS at TMP = - - 5 kPa assesses the rigidity of the tracheal cartilage rings and the tendency of the tracheal muscle to protrude into the tracheal lumen. The collapse of the tracheal soft tissue has been shown to be reduced by hormone- or neurally-induced contraction of the tracheal smooth muscle (Olsen et al 1967, Coburn et al 1972, Knudson and Knudson 1975): although the force of contraction of the trachealis muscle is primarily circumferential, this contraction decreases the diameter of the trachea and forms a cartilaginous tube with a decreased compressibility. Knudson and Knudson (1975) and Bouhuys and Van de Woestijne (1971) have demonstrated that tracheal or bronchial muscle contraction can result in decreased flow conductance at low flows but increased flow rates at higher driving pressures when the less compliant trachea resists flow-limiting collapse. Because the hormonal response to exercise is mainly sympathetic, ie, increased plasma epinephrine and norepinephrine (Thornton 1985), it certainly does not induce any tracheal smooth muscle contraction. Moreover, previous studies have demonstrated that maximal or normal inflation of the lungs can temporarily abolish or reduce airway smooth muscle tone (Nadel and Tierney 1961, Drazen et al 1979, Gunst and Lai-Fook 1983). Pride et al (1967) and Knudson and Knudson (1975) pointed out that, once the airways collapse begins, the Bernoulli effect (Knudson and Knudson 1975) would begin to contribute, to a progressively increasing degree, to further diminution in airway X-SA. The changes in X-SA observed in the present work were simultaneous with dramatic changes in its shape: as there was no overlapping of the cartilage ends, compressive TMP results in a concave-convex X-SA (Fig 2). If the same is true in vivo, when the trachea is submitted to a compressive TMP, this change of shape might induce an increase in resistance by increasing frictional resistance and by promoting turbulence. The authors concluded that the large extmthoracic and intrathoracic airways are sufficiently compliant to be collapsible when submitted to high, but nevertheless physiological, compressive TMP and therefore that this in vitro study confirms the hypothesis previously suggested that the increase in total pulmonary resistance recorded in horses during strenuous exercise may be due to a partial collapse of the large airways.
T. A r t , P. L e k e u x
60 Acknowledgements
The authors wish to thank M. Delacroix for her helpful technical assistance and M. Leblond for typing the manuscript. We also thank Professor G. Maghuin-Rogister and G. Degand for their valuable advice and help with measuring the ATP content. References ART, T., SERTEYN, D. & LEKEUX, P. (1988) Effect of exercise on the partitioning of equine respiratory resistance. Equine Veterinary Journal 20, 268-273 ART, T., ANDERSON, L. S., ROBERTS, C. A., WOAKES, A. J., BUTLER, P. J., SNOW, D. H. & LEKEUX, P. (1990) Mechanics of breathing during strenuous exercise in thoroughbred horses. Respiration Physiology 82, 279-284 BHUTANI, V. K., RUBENSTEIN, S. D. & SHAFPER, T. H. (1981) Pressure-volume relationships of trachea in fetal newborn and adult rabbits. Respiration Physiology 41,221-231 BOBBAERS, H., CLEMENT, J. & VAN DE WOESTIJNE, K. P. (1978) Dynamic yiscoelastic properties of the canine trachea.
Journal of Applied Physiology: Respiration Environment and Exercise Physiology 44, 137-143 BOUHUYS, A. & VAN DE WOESTIJNE, K. P. (1971) Mechanical consequences of airway smooth muscle relaxation. Journal of Applied Physiology 30, 670-676 BROWN, I. G., WEBSTER, P. M., ZAMEL, N. & HOFFSTEIN, V. (1986) Changes in tracheal cross-sectional area during MueUer and Valsalva maneuvers in humans. Journal of Applied Physiology 60, 1865-1870 COBURN, R. F., THORNTON, D. & ARTS, R. (1972) Effect of trachealis muscle contraction on tracheal resistance to airflow. Journal of Applied Physiology 32, 397-403 CROTEAU, J. R. & COOK, C. D. (1961) Volume-pressure and length-tension measurements in human tracheal and bronchial segments. Journal of Applied Physiology 16, 170-172 DEKKER, E. & LEDEBOER, R. (1961) Compression of the tracheobronchial tree by the action of the voluntary respiratory musculature in normal individuals a n d in patients with asthma and emphysema. American Journal of Roentgenology, Radium Therapy and Nuclear Medicine 85, 217-228 DRAZEN, J. M., LORING, S. H., JACKSON, A. C., SNAPPER, J. R. & INGRAM, R. H. (1979) Effects of volume history on airway changes induced by histamine or vagal stimulation. Journal
GRISCOM, N. T. & WOHL, M. E. B. (1983) Tracheal size and shape: effects of change in intraluminal pressure. Radiology 149, 27-30 GUNST, S. J. & LAI-FOOK, S. J. (1983) Effect of inflation on trachealis muscle tone in canine tracheal segments in vitro. Journal
of Applied Physiology: Respiration, Environment and Exercise Physiology 54, 906-913 HOFFSTEIN, V., GLASS, G. M., WOHL, M. E., DORKIN, H. L., STRIEDER, D. J. & FREDBERG, J. J. (1980) Tracheal geometry and compliance during breathing. Physiologist 23, 165 KNUDSON, R. J. & KNUDSON, D. E. (1975) Effect of muscle constriction on flow-limiting collapse of isolated canine trachea. Journal of Applied Physiology 38, 125-131 MARTIN, B. H. & PROCTOR, D. F. (1958) Pressure-volume measurements on dog bronchi. Journal of Applied Physiology 13, 337-343 MISEROCCHI, G. & AGOSTONI, E. (1973) Longitudinal forces acting on the trachea. Respiration Physiology 17, 62-71 MORENO, R. H., McCORMACK, G. S., BRENDAN, J., MULLEN, M., HOGG, J. C., BERT, J. & PARE, P. D. (1986) Effect of intravenous papain on tracheal pressure-volume curves in rabbits. Journal of Applied Physiology 60, 247-252 NADEL, J. A. & TIERNEY, E. (1961) Effect of a previous deep inspiration on airway resistance in man. Journal of Applied Physiology 16, 717-719 OLSEN, C. R., STEVENS, A. E. & MclLROY, M. B. (1967) Rigidity of trachea and bronchi during muscular constriction. Journal of Applied Physiology 23, 27- 34 PRIDE, N. B., PERMUTT, S., RILEY, R. L. & BROMBERGERBARNEA, B. (1967) Determinants of maximum expiratory flow from the lungs. Journal of Applied Physiology 23, 646-662 SOUHRADA, J. F. & DICKEY, D. W. (1976) Mechanical activities of trachea as measured in vitro and in vivo. Respiration Physiology 26, 27-40 TANDLER, B., SHERMAN, J. M., BOAT, T. F. & WOOD, R. E. (1983) Surface architecture of the mucosal epithelium of the cat trachea: II. Structure and dynamics of the membranous portion. American Journal of Anatomy 168, 133-144 THORNTON, J. R. (1985) Hormonal responses to exercise and training. VeterinaryClinics of North America, Equine Practice 1, 477-498 WITTENBORG, M. H., GYEPES, M. T. & CROCKER, D. (1967) Tracheal dynamics in infants with respiratory distress, stridor, and collapsing trachea. Radiology 88, 653-662
of Applied Physiology: Respiration Environment and Exercise Physiology 47, 657-665 GILLESPIE, J. R. (1974) The role of the respiratory system during exertion. Journal of the South African VeterinaryAssociation 45, 305-309
Received July 11, 1990 Accepted January 21, 1991
