Low-frequency pulmonary impedance in rabbits and its response to inhaled methacholine R. TEPPER, Meakins-Christie
J. SATO,
B. SUKI,
Laboratories,
TEPPER, R., J. SATO, B. SUKI, BATES. Low-frequency pulmonary
J. G. MARTIN, impedance
J. G. MARTIN,
McGill AND
University, J. H. T.
in rabbits and its
methacholine. J. Appl. Physiol. 73(l): 290295, 1992.-We assessedpulmonary mechanics in six openchest rabbits (3 young and 3 adult) by the forced oscillation technique between 0.16 and 10.64 Hz. Under control conditions, pulmonary resistance(RL) decreasedmarkedly between 0.16 and 4 Hz, after which it becamereasonablyconstant. Measurementsof alveolar pressure from two alveolar capsulesin each rabbit showedthat the large decreaseof RL with increasing frequency below 4 Hz was due to lung tissue rheology and that tissueresistancewascloseto zero above 4 Hz. Estimates of resistanceand elastance,also obtained by fitting tidal ventilation data at 1 Hz to the equation of the linear single-compartment model, gave values for RL motion that were slightly higher than those obtained by forced oscillations at the same frequency, presumably becauseof the flow dependenceof airways resistance.After treatment with increasingdosesof aerosolized methacholine, RL and pulmonary elastance between 0.16 and 1.34 Hz progressively increased, as did the point at which the pulmonary reactance crossedzero (the resonant frequency). The alveolar pressuremeasurementsshowedthe lung to becomeincreasingly inhomogeneouslyventilated in all six animals,whereasin the three younger rabbits lobar atelectasis developedat high methacholine concentrations and the alveolar capsulesceasedto communicate with the central airways. We conclude that the low-frequency pulmonary impedanceof rabbits exhibits the samequalitative features observedin other speciesand that it is a sensitive indicator of the changesin pulmonary mechanicsoccurring during bronchoconstriction.
response to inhaled
AND
Montreal,
J. H. T. BATES Quebec H2X
2P2, Canada
limited
to humans (11, 21), dogs (3-5, 10, 17), and rats (12). However, a number of other species serve as useful
animal models for the study of both normal lung function and pulmonary pathophysiology. An important example is the rabbit, which is frequently used in investigations of neonatal lung disease (14), acute lung injury (8,22), and airway reactivity (9, 18, 23). To date, such studies have been limited by the fact that pulmonary mechanics are invariably assessed at only a single frequency. Although the pulmonary impedance in rabbits has been investigated by Sullivan et al. (22), they did not study frequencies below 4 Hz, where most of the negative frequency dependence of resistance in other species is found. The purpose of the present study was therefore to investigate pulmonary impedance at low frequencies in rabbits under normal conditions and after bronchoconstriction. The rationale for this was twofold: 1) to confirm that the rabbit has low-frequency pulmonary and tissue impedances exhibiting features similar to those observed in other species and 2) to evaluate the extent of the changes in these impedances occurring with acute bronchoconstriction. METHODS
Animal preparation and instrumentation. Six male New Zealand White rabbits were evaluated: three were 6 mo old (3.0-3.5 kg), and three were 1 mo old (0.5-0.9 kg). Animals were anesthetized with pentobarbital sodium airways resistance; forced oscillation technique; tissue resis- (30-50 mg/kg iv) via a marginal ear vein and placed in tance; pulmonary elastance the supine position. A tracheostomy was performed below the larynx. A short snugly fitting cannula was inserted into the trachea and tied with surgical thread. After paralysis with pancuronium bromide (0.1-0.3 mg/ THE MECHANICAL IMPEDANCE of the lungs at low frequencies has been the focus of considerable interest in kg), animals were mechanically ventilated (Harvard recent years. The most striking observation responsible small animal ventilator 683) with a tidal volume of 5 ml/ for this interest is the huge decrease in resistance with kg at a rate of 60 cycles/min. increasing frequency that occurs below 2 Hz (3-5,10-12, Tracheal airflow (V) was measured with a pneumota17, 21). Because spontaneous breathing frequencies are chometer and a differential piezoresistive pressure transalso 0.98 in all cases with the following exceptions due to interference by cardiogenic oscillations: rabbit 2, 4.24 and 4.88 Hz at all doses of MCh; rabbit 3, 4.88, 6.32, and 10.64 Hz at baseline and 4.88 and 10.64 Hz at 32 mglml; rabbit 4, 3.76 Hz at baseline; rabbit 5, 3.76 and 4.88 Hz at baseline and 4.88 Hz at 64 mg/ml. In rabbit 6, for some reason the coherence values at baseline and at 16 and 32 mg/ml were lower than the others (mean 0.94), whereas at 64 mg/ml they were all >0.99. Table 1 contains the estimates of RL(~), Rti(l), and Raw(l), calculated by linearly interpolating between the forced oscillation values obtained at 0.88 and 1,36 Hz. Also shown are the values of RL(mv), Rti(mv), and Raw(mv) obtained during mechanical ventilation at 1 Hz (Eq. 5). All values in each animal have been normalized to the corresponding value of AL. The two determinations of Rti were similar, whereas both RL(mv) and Raw(mv) were higher than AL and Raw(l). Table 2 contains the estimates of EL(~), EL(mv), Eti(l), and Eti(mv). EL(~) and Eti(1) are lower than EL(mv) and Eti(mv). However, within either technique EL and Eti are the same.
l
RL( f) at selected MCh concentrations are shown in Fig. 2. In general, in all six rabbits, RL( f) increased with increasing MCh concentration. RL( f) also continued to show a marked decrease with f < 4 Hz. Increasing concentrations of MCh also generally resulted in a decrease in XL( f ) and an increase in the resonant f of the system, as indicated by the fat which XL(~) crossed zero (Fig. 3). Figure 4 shows EL(~) between 0.16 and 1.36 Hz at the same concentrations of MCh used in Fig. 2. EL(~) shows a slight increase with f, particularly at the higher MCh concentrations. At any particular f, it also increased markedly with increasing MCh dose. Under baseline conditions, the two PA signals measured during tidal ventilation were identical by inspection in five of six rabbits, suggesting that the lungs of these rabbits were functioning homogeneously at 1 Hz. In one (immature) rabbit, there was a small phase difference visually discernible between the two PA values at 1 Hz that became more evident at higher f, where the Rti( f ) became negative. During bronchoconstriction, the two PA values measured in all the rabbits became disparate to variable degrees, indicating the onset of heterogeneous ventilation within the lungs. This was accompanied by variable responses in Rti( f ) between and within animals. In one rabbit the Rti( f) obtained with both PA values became negative at high MCh concentrations. In two rabbits one Rti( f) became negative while the other remained positive. In the remaining three rabbits, both Rti( f) values remained positive. Also, the degree of inTABLE 2. Lung and lung tissue elastances obtained from forced oscillation and tidal ventilation data Rabbit
No.
EL(~)
1 2 3 4 5 6
1.0 1.0 1.0 1.0 1.0 1.0
Mean tSD
1.0
EL( mv) 1.7 1.3 1.3 1.2 1.4 1.6 1.4
to.2
Eti( 1) 1.1 1.0 1.0 1.0 1.0 1.0 1.0
to.0
Eti( mv) 1.8 1.4 1.4 1.2 1.5 1.6 1.3
to.3
Values are expressed as kPa/l. EL and Eti, lung and lung tissue elastance. In each rabbit, values were normalized with respect to corresponding EL( 1).
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
LOW-FREQUENCY
IMPEDANCE
IN
0,’
n
0.0 f
32,64, and 128 mg/ml (remaining 3 lines from bottom to
homogeneity between the two capsules increasedprogressively with the dose of MCh. Under baseline conditions, the mean difference between the two Rti values obtained at the lowest frequency of 0.16 Hz was 4.8% of the mean value. This increased to 27.6, 61.9, and 103.0% as the MCh dose increased to 32, 64, and 128 mg/ml, respectively. The corresponding mean values of the percent dif-
f (Hz) 2
4
6
8’0
1.0
1.5 f
top)*
0
00 0.5
(Hz)
RL( f) vs. f in each of 6 rabbits under control conditions (bottom line in each panel) and RL(f) with increasing doses of methaFIG. 2.
choline (MCh):
293
RABBITS
0246
8
10
0.0
0.5
1.0
1.5
0-w
4. EL(~) vs. f in each of 6 rabbits under control conditions (bottom line in each panel) and EL(f ) obtained with increasing doses of MCh (see Fig. 2). EL, pulmonary elastance. FIG.
ferences in Xti(0.16) were 0.9,15.4,45.6, and 96.5 at baseline and MCh concentrations of 32, 64, and 128 mg/ml, respectively. As an example of the type of disparity between the two capsules that we encountered, Fig. 5 shows the Rti( f) obtained from each of the two PA values in rabbit 3 under control conditions and at two concentrations of MCh. One of the Rti( f ) values increasedprogressively with MCh, whereas the other increased at the low concentrations and then became almost zero at higher concentrations at f > 2 Hz. In rabbits 4-6 at MCh concentrations 264 mg/ml, the alveolar regions under the two capsules ceased to communicate with the central airways, as evidenced by the cessation of the usual breath-by-breath oscillations in PA with mechanical ventilation. This phenomenon was always followed by development of visible atelectasis of the lobes on which the capsules were placed. It also seemed to be associated with a larger increase in RL( f) than occurred at the lower MCh concentrations (Fig. 2, rabbits 4-6). After completion of the MCh challenge, the atelectatic lobes reexpanded and the two PA values regained their former breath-by-breath oscillations in phase with ventilation. DISCUSSION
Our results demonstrate
that under control conditions
ZL exhibits the same features in rabbits as in humans (11, 21), dogs (3-5, IO), and rats (12). In particular, RL(~)
FIG. 3. XL(f ) vs. fin each of 6 rabbits under control conditions (top line in each panel) together with XL(~) with increasing doses of MCh (see Fig. 2). XL, pulmonary reactance.
decreased markedly between 0.16 and 4 Hz. From 4 and 10.64 Hz, RL( f) remained relatively constant, similar to the results of Sullivan et al. (22). Our measurements of PA also showed that Raw(f) was essentially constant over the entire f range, thereby demonstrating that the low-frequency variation in RL( f ) was due to the rheological properties of lung tissue, as has been shown in normal
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
294
LOW-FREQUENCY
IMPEDANCE
#3
#3 a I I
i
0
2
4
6
8
10
f (Hz)
5. Rti( f) vs. f from each of the 2 alveolar pressures measured in 3 under control conditions (solid line) and with 16 (dash-dot line) and 64 mg/ml MCh (dashed line). FIG. rabbit
dogs (10). In addition, we found that Rti( f ) became close to zero above 4 Hz, similar to dogs (3). This has practical significance for the study of airway phenomena in rabbits, because it means that Raw(f) is given by RL( f) at f > 4 Hz. In contrast, at lower f, RL( f ) reflects a variable degree of Rti( f), comprising as much as 88% of the former at 0.16 Hz. We note also that our results apply to both the older group of rabbits ( 1-3) and the younger group (4-6). 0 f course, with only three animals in each group, we cannot draw strong conclusion .s about the efNevertheless the only difference befects of maturity tween the younger and older rabbits apparent from Fig. 2 is that the values of RL( f) are considerably higher in the younger rabbits as a consequence of their smaller sizes. The values of RL(mv) were somewhat larger than the corresponding values of RL( l), RL,, being on average 130% of Rx,(l) (Table 1). These differences are relatively small compared with the variation in RL( f) occurring between 0.16 and 4 Hz (Figs. 1 and 3) and probably reflect some nonlinear aspect of the respiratory system. Our results indicate that the most important nonlinearity is the flow dependence of Raw, because Raw(l) was smaller than Raw(mv), whereas there was no difference between Rti( 1) and Rti(mv) (Table 1). One might have expected there also to have been differences between RL( 1) and RL(mv) because of the differences in mean
IN
RABBITS
lung vol .ume betwee n the forced oscillation and tidal ventilation tech .niques. However, this should have resulted in differences between Rti( 1) and Rti(mv), because tissue viscance has been reported to be volume dependent (15). Presumably the conditions of our experiment were such that volume-dependent effects were small, in contrast, for example, to the study of Lutchen and Jackson (17), who found a tidal volume dependence of the tissues when oscillating around functional residual capacity in dogs. The progressive heterogen .eity of alveolar ventilation th .at became apparent in the rabbits exposed to increasing doses of MCh (see RESULTS and Fii. 5) is similar to that reported in dogs after inhalation of histamine (16) and presumably refl .ected a combination of inhomogeneous aerosol deposition and inhomogeneous responsiveness throughout was the lun g. This inhomogeneity also presumably resp ionsible for the occurrence of physically- meaningless negative values of Rti( f ). Indeed, Fredberg et al. (7) also found negative Rti values after histamine exposure in dogs, which they suggested was due to an airway shunt compliance. Their theory might also explain our own results. Alternatively, our negative Rti may have resulted from parallel inhomogeneities across the lung. That is, we used the V measured at the trachea to calculate regional Rti in an inhomogeneous lung, whereas in reality the V entering each alveolar region was not proportional to tracheal V. Regional V must be a function of the mechanical properties of the region concerned compared with those of other regions that compete for the total V. In any case, compartmentalization of thea lung in either a parallel or serial 1fashion means that V at the trachea does not reflect V at the alveolar level, which can potentially result in the calculation of a negative Rti. We also found variable relative responses to MCh at the low and high ends of the frequency-spectrum studied, as can be discerned in Fig. 2. The ratio of R~(0.16) to R~(10.64) had an average value of 6.08 t 3.21 under control conditions. At the highest doses of MCh represented in Fig. 2, this ratio had changed by factors of 0.58, 1.63, 0.43, 2.18, 1.18, and 1.20 for rabbits 1, 2, 3, 4, 5, and 6, respectively. Thus some rabbits experienced a greater relative increase in RL( f) at low frequencies, reflecting predominantly tissue viscoelastic properties, whereas others had a greater change at high frequencies where the resistance of the airways is of much greater importance. Such variability with MCh has been noted elsewhere in dogs (17) and rabbits (19). The three less mature rabbits developed lobar atelectasis during the MCh c hallenge. Interestingly, this suggests that age may be an importan t factor in the response of rabbits to MCh. This is consistent with in vitro-findings of greater airway smooth muscle contractility in immature rabbits (13, 23) and pigs (20) than in their mature counterparts. Lobar atelectasis during bronchial challenge has also not been reported in mature dogs instrumented with alveolar capsules. Although the occurrence of lobar atelectasis obliterated our PA signals and so prevented assessment of regional tissue properties at high MCh concentrations, the RL( f) measurements continued to indicate overall changes in lung mechanics in an expected manner (Figs. 2-4). For example, we noticed that the occurrence of atel-
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
LOW-FREQUENCY
IMPEDANCE
ectasis in the three younger rabbits (4-6) tended to be accompanied by a particularly large increase in RL( f) at the higher f (Fig. 4). We suspect that this can be explained in part by the fact that atelectasis suddenly reduces the total volume of the lung and consequently increases its resistance simply due to loss of available airways. An increase in elastance due to loss of available lung volume should accompany this atelectasis, and indeed we noticed that EL(~) below 1.36 Hz (Fig. 3) also shows the same tendency to increase more markedly with the occurrence of atelectasis. Finally, although the data obtained in the present study pertain to the lung alone, we can speculate on how our results might carry over to the intact rabbit in which tracheal pressure-flow relationships are also influenced by the chest wall. The tissues of the chest wall are highly viscoelastic and so contribute significantly to respiratory impedance at low frequencies (1, 3). Also, the chest wall has been shown in dogs to have a component that behaves similarly to Raw, as determined by the flow interruption technique (2, 3), which would contribute to respiratory impedance at frequencies > 5 Hz, where viscoelastic dissipation of energy no longer occurs. It is quite likely, then, that total respiratory impedance in rabbits would exhibit noticeable differences compared with ZL alone. However, given that bronchoconstriction presumably affects the lung exclusively, we would expect that the differences between respiratory impedance and ZL would become relatively less important as bronchoconstriction proceeds. For the same reason, changes in lung mechanics could be accurately inferred from changes in total respiratory mechanics. In summary, we have found that the forced oscillation technique provides a convenient and informative way of characterizing pulmonary mechanics in rabbits under control conditions and during severe bronchoconstriction. Furthermore the ZL and Zti spectra obtained show that the mechanical properties of the rabbit lung are qualitatively similar to those of other species. In particular, we found that the f range we used is useful for assessing pulmonary mechanics, because it provides information pertaining to the lung tissues at f < 4 Hz while, at least under control conditions, reflecting exclusively airway properties above 4 Hz. Although we cannot be sure that< the same is true during bronchoconstriction because of the lung inhomogeneities induced, if Rti( f) also falls to zero above 4 Hz with MCh, then RL( f) above this fcontinues to be a measure of overall Raw. This work was supported by the Medical Research Council of Canada (MRC) and the EL/JTC Memorial Research Fund. R. Tepper was supported by The American Lung Association of Indiana; J. G. Martin is a Scientist of the MRC; J. H. T. Bates was a Scholar of the MRC; and J. Sato was supported by Chiba University Hospital, Chiba, Japan. Address for reprint requests: J. H. T. Bates, Meakins-Christie Laboratories, 3626 St. Urbain St., Montreal, Quebec H2X 2P2, Canada. Received 18 January 1991; accepted in final form 27 February 1992. REFERENCES 1. BARNAS, G. M., K. YOSHINO, S. H. LORING, AND J. MEAD. Impedance and relative displacements of relaxed chest wall up to 4 Hz. J. Appl. Physiol.
62: 71-81,
1987.
IN RABBITS
295
J. H. T., T. ABE, P. V. ROMERO, AND J. SATO. Measurement of alveolar pressure in closed-chest dogs during flow interruption. J. Appl. Physiol. 67: 488-492, 1989. BATES, J. H. T., K. A. BROWN, AND T. KOCHI. Respiratory mechanics in the normal dog determined by expiratory flow interruption. J. Appl. Physiol. 67: 2276-2285, 1989. BATES, J. H. T., F. SHARDONOFSKY, AND D. E. STEWART. The low-frequency dependence of respiratory system resistance and elastance in normal dogs. Respir. Physiol. 78: 369-382, 1989. BRUSASCO, V., D. 0. WARNER, K. C. BECK, J. R. RODARTE, AND K. REHDER. Partitioning of pulmonary resistance in dogs: effect of tidal volume and frequency. J. Appl. Physiol. 66: 1190-1196, 1989. DAROCZY, B., AND Z. HANTOS. Generation of optimum pseudorandom signals for respiratory impedance measurements. 1nt. J.
2. BATES,
3.
4.
5.
6.
Biomed. Comput. 25: 21-31,199O. 7. FREDBERG, J. J., R. H. INGRAM, J. M. DRAZEN. Nonhomogeneity
R. G. CASTILE, G. M. GLASS, AND of lung response to inhaled histamine assessed with alveolar capsules. J. Appl. Physiol. 58: 1914-
1922,1985. 8. GILLIARD,
N., P. M. RICHMAN, T. A. MERRITT, AND R. G. SPRAGG. Effect of volume and dose on the pulmonary distribution of exogenous surfactant administered to normal rabbits or to rabbits with oleic acid lung injury. Am. Rev. Respir. Dis. 141: 743-747, 1990. 9. HABIB, M. P., A. M. DUNN, R. E. SOBONYA, C. C. BAUMGARTERRER, J. D. NEWELL, AND M. HALONEN. Immunoglobulin E anaphylaxis in rabbits: mechanisms of pulmonary resistance and compliance. J. Appl. Physiol. 64: 1009-1016, 1988. 10. HANTOS, Z., B. DAROCZY, T. CSENDES, B. SUKI, AND S. NAGY. Modeling of low-frequency pulmonary impedance in dogs. J. Appl. Physiol. 68: 849-860, 1990. 11. HANTOS, Z., B. DAROCZY,
B. SUKI, G. GALGOCZY, AND T. CSENDES. Forced oscillatory impedance of the respiratory system at low frequencies. J. Appl. Physiol. 60: 123-132, 1986. 12. HANTOS, Z., B. DAROCZY, B. SUKI, AND S. NAGY. Low-frequency respiratory mechanical impedance in the rat. J. Appl. Physiol. 63: 36-43,1987. 13. HAYASHI,
S., AND T. NOBORU. Age related alterations in the response of rabbit tracheal smooth muscle to agents. J. Pharmacol.
Exp. Ther. 214: 675-681, 1980. 14. IKEGAMI, M., D. BERRY, T. EL KADY, A. PETTENAZZO, AND A. JOBE. Corticosteroids and surfactant change
S. SEIDNER,
lung function and protein leaks in the lungs of ventilated premature rabbits. J.
Clin. Invest. 79: 1371-1378, 1987. 15. KARIYA, S. T., L. M. THOMPSON, E. P. INGENITO, AND R. H. INGRAM. Effects of lung volume, volume history, and methacholine on lung tissue viscance. J. Appl. Physiol. 66: 977-982, 1989. 16. LUDWIG, M. S., P. V. ROMERO, P. D. SLY, J. J. FREDBERG, AND J. H. T. BATES. Interpretation of interrupter resistance after histamine-induced constriction in the dog. J. Appl. Physiol. 68: 16511656,199O. 17. LUTCHEN, K. R., AND A. C. JACKSON. Effects of tidal volume and
methacholine on low-frequency total respiratory impedance in dogs. J. Appl. Physiol. 68: 2128-2138, 1990. 18. METZGER, W. J. Late phase asthma in an allergic rabbit model. In: Handbook of Late Phase Reactions, edited by W. Dorsch. Boca Raton, FL: CRC, 1990, p. 347-362. 19. ROMERO, P. V., AND M. S. LUDWIG. Maximal methacholine-induced constriction in rabbit lung: interactions between airways and tissue? J. Appl. Physiol. 70: 1044-1050, 1991. 20. SPARROW, M., AND H. MITCHELL. Contraction of smooth muscle of pig airway tissues from before birth to maturity. J. Appl. Physiol. 68: 468-477,199O. 21. SUKI, B., R. PESLIN,
C. DWIVIER, AND R. FARRE. Lung impedance in healthy humans measured by forced oscillations from 0.01 to 0.1 Hz. J. Appl. Physiol. 67: 1623-1629, 1989. 22. SULLIVAN, K. J., M. DURAND, T. YE, AND H. K. CHANG. Evaluation of lung mechanics in rabbits using short duration flow pulses. Am. Rev. Respir. Dis. 140: 17-24, 1989. 23. TANAKA, D., AND M. GRUNSTEIN.
Maturation of neuromodulatory effect of substance P in rabbit airways. J. Clin. Invest. 85: 345-350, 1990.
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
J. SATO,
B. SUKI,
Laboratories,
TEPPER, R., J. SATO, B. SUKI, BATES. Low-frequency pulmonary
J. G. MARTIN, impedance
J. G. MARTIN,
McGill AND
University, J. H. T.
in rabbits and its
methacholine. J. Appl. Physiol. 73(l): 290295, 1992.-We assessedpulmonary mechanics in six openchest rabbits (3 young and 3 adult) by the forced oscillation technique between 0.16 and 10.64 Hz. Under control conditions, pulmonary resistance(RL) decreasedmarkedly between 0.16 and 4 Hz, after which it becamereasonablyconstant. Measurementsof alveolar pressure from two alveolar capsulesin each rabbit showedthat the large decreaseof RL with increasing frequency below 4 Hz was due to lung tissue rheology and that tissueresistancewascloseto zero above 4 Hz. Estimates of resistanceand elastance,also obtained by fitting tidal ventilation data at 1 Hz to the equation of the linear single-compartment model, gave values for RL motion that were slightly higher than those obtained by forced oscillations at the same frequency, presumably becauseof the flow dependenceof airways resistance.After treatment with increasingdosesof aerosolized methacholine, RL and pulmonary elastance between 0.16 and 1.34 Hz progressively increased, as did the point at which the pulmonary reactance crossedzero (the resonant frequency). The alveolar pressuremeasurementsshowedthe lung to becomeincreasingly inhomogeneouslyventilated in all six animals,whereasin the three younger rabbits lobar atelectasis developedat high methacholine concentrations and the alveolar capsulesceasedto communicate with the central airways. We conclude that the low-frequency pulmonary impedanceof rabbits exhibits the samequalitative features observedin other speciesand that it is a sensitive indicator of the changesin pulmonary mechanicsoccurring during bronchoconstriction.
response to inhaled
AND
Montreal,
J. H. T. BATES Quebec H2X
2P2, Canada
limited
to humans (11, 21), dogs (3-5, 10, 17), and rats (12). However, a number of other species serve as useful
animal models for the study of both normal lung function and pulmonary pathophysiology. An important example is the rabbit, which is frequently used in investigations of neonatal lung disease (14), acute lung injury (8,22), and airway reactivity (9, 18, 23). To date, such studies have been limited by the fact that pulmonary mechanics are invariably assessed at only a single frequency. Although the pulmonary impedance in rabbits has been investigated by Sullivan et al. (22), they did not study frequencies below 4 Hz, where most of the negative frequency dependence of resistance in other species is found. The purpose of the present study was therefore to investigate pulmonary impedance at low frequencies in rabbits under normal conditions and after bronchoconstriction. The rationale for this was twofold: 1) to confirm that the rabbit has low-frequency pulmonary and tissue impedances exhibiting features similar to those observed in other species and 2) to evaluate the extent of the changes in these impedances occurring with acute bronchoconstriction. METHODS
Animal preparation and instrumentation. Six male New Zealand White rabbits were evaluated: three were 6 mo old (3.0-3.5 kg), and three were 1 mo old (0.5-0.9 kg). Animals were anesthetized with pentobarbital sodium airways resistance; forced oscillation technique; tissue resis- (30-50 mg/kg iv) via a marginal ear vein and placed in tance; pulmonary elastance the supine position. A tracheostomy was performed below the larynx. A short snugly fitting cannula was inserted into the trachea and tied with surgical thread. After paralysis with pancuronium bromide (0.1-0.3 mg/ THE MECHANICAL IMPEDANCE of the lungs at low frequencies has been the focus of considerable interest in kg), animals were mechanically ventilated (Harvard recent years. The most striking observation responsible small animal ventilator 683) with a tidal volume of 5 ml/ for this interest is the huge decrease in resistance with kg at a rate of 60 cycles/min. increasing frequency that occurs below 2 Hz (3-5,10-12, Tracheal airflow (V) was measured with a pneumota17, 21). Because spontaneous breathing frequencies are chometer and a differential piezoresistive pressure transalso 0.98 in all cases with the following exceptions due to interference by cardiogenic oscillations: rabbit 2, 4.24 and 4.88 Hz at all doses of MCh; rabbit 3, 4.88, 6.32, and 10.64 Hz at baseline and 4.88 and 10.64 Hz at 32 mglml; rabbit 4, 3.76 Hz at baseline; rabbit 5, 3.76 and 4.88 Hz at baseline and 4.88 Hz at 64 mg/ml. In rabbit 6, for some reason the coherence values at baseline and at 16 and 32 mg/ml were lower than the others (mean 0.94), whereas at 64 mg/ml they were all >0.99. Table 1 contains the estimates of RL(~), Rti(l), and Raw(l), calculated by linearly interpolating between the forced oscillation values obtained at 0.88 and 1,36 Hz. Also shown are the values of RL(mv), Rti(mv), and Raw(mv) obtained during mechanical ventilation at 1 Hz (Eq. 5). All values in each animal have been normalized to the corresponding value of AL. The two determinations of Rti were similar, whereas both RL(mv) and Raw(mv) were higher than AL and Raw(l). Table 2 contains the estimates of EL(~), EL(mv), Eti(l), and Eti(mv). EL(~) and Eti(1) are lower than EL(mv) and Eti(mv). However, within either technique EL and Eti are the same.
l
RL( f) at selected MCh concentrations are shown in Fig. 2. In general, in all six rabbits, RL( f) increased with increasing MCh concentration. RL( f) also continued to show a marked decrease with f < 4 Hz. Increasing concentrations of MCh also generally resulted in a decrease in XL( f ) and an increase in the resonant f of the system, as indicated by the fat which XL(~) crossed zero (Fig. 3). Figure 4 shows EL(~) between 0.16 and 1.36 Hz at the same concentrations of MCh used in Fig. 2. EL(~) shows a slight increase with f, particularly at the higher MCh concentrations. At any particular f, it also increased markedly with increasing MCh dose. Under baseline conditions, the two PA signals measured during tidal ventilation were identical by inspection in five of six rabbits, suggesting that the lungs of these rabbits were functioning homogeneously at 1 Hz. In one (immature) rabbit, there was a small phase difference visually discernible between the two PA values at 1 Hz that became more evident at higher f, where the Rti( f ) became negative. During bronchoconstriction, the two PA values measured in all the rabbits became disparate to variable degrees, indicating the onset of heterogeneous ventilation within the lungs. This was accompanied by variable responses in Rti( f ) between and within animals. In one rabbit the Rti( f) obtained with both PA values became negative at high MCh concentrations. In two rabbits one Rti( f) became negative while the other remained positive. In the remaining three rabbits, both Rti( f) values remained positive. Also, the degree of inTABLE 2. Lung and lung tissue elastances obtained from forced oscillation and tidal ventilation data Rabbit
No.
EL(~)
1 2 3 4 5 6
1.0 1.0 1.0 1.0 1.0 1.0
Mean tSD
1.0
EL( mv) 1.7 1.3 1.3 1.2 1.4 1.6 1.4
to.2
Eti( 1) 1.1 1.0 1.0 1.0 1.0 1.0 1.0
to.0
Eti( mv) 1.8 1.4 1.4 1.2 1.5 1.6 1.3
to.3
Values are expressed as kPa/l. EL and Eti, lung and lung tissue elastance. In each rabbit, values were normalized with respect to corresponding EL( 1).
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
LOW-FREQUENCY
IMPEDANCE
IN
0,’
n
0.0 f
32,64, and 128 mg/ml (remaining 3 lines from bottom to
homogeneity between the two capsules increasedprogressively with the dose of MCh. Under baseline conditions, the mean difference between the two Rti values obtained at the lowest frequency of 0.16 Hz was 4.8% of the mean value. This increased to 27.6, 61.9, and 103.0% as the MCh dose increased to 32, 64, and 128 mg/ml, respectively. The corresponding mean values of the percent dif-
f (Hz) 2
4
6
8’0
1.0
1.5 f
top)*
0
00 0.5
(Hz)
RL( f) vs. f in each of 6 rabbits under control conditions (bottom line in each panel) and RL(f) with increasing doses of methaFIG. 2.
choline (MCh):
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10
0.0
0.5
1.0
1.5
0-w
4. EL(~) vs. f in each of 6 rabbits under control conditions (bottom line in each panel) and EL(f ) obtained with increasing doses of MCh (see Fig. 2). EL, pulmonary elastance. FIG.
ferences in Xti(0.16) were 0.9,15.4,45.6, and 96.5 at baseline and MCh concentrations of 32, 64, and 128 mg/ml, respectively. As an example of the type of disparity between the two capsules that we encountered, Fig. 5 shows the Rti( f) obtained from each of the two PA values in rabbit 3 under control conditions and at two concentrations of MCh. One of the Rti( f ) values increasedprogressively with MCh, whereas the other increased at the low concentrations and then became almost zero at higher concentrations at f > 2 Hz. In rabbits 4-6 at MCh concentrations 264 mg/ml, the alveolar regions under the two capsules ceased to communicate with the central airways, as evidenced by the cessation of the usual breath-by-breath oscillations in PA with mechanical ventilation. This phenomenon was always followed by development of visible atelectasis of the lobes on which the capsules were placed. It also seemed to be associated with a larger increase in RL( f) than occurred at the lower MCh concentrations (Fig. 2, rabbits 4-6). After completion of the MCh challenge, the atelectatic lobes reexpanded and the two PA values regained their former breath-by-breath oscillations in phase with ventilation. DISCUSSION
Our results demonstrate
that under control conditions
ZL exhibits the same features in rabbits as in humans (11, 21), dogs (3-5, IO), and rats (12). In particular, RL(~)
FIG. 3. XL(f ) vs. fin each of 6 rabbits under control conditions (top line in each panel) together with XL(~) with increasing doses of MCh (see Fig. 2). XL, pulmonary reactance.
decreased markedly between 0.16 and 4 Hz. From 4 and 10.64 Hz, RL( f) remained relatively constant, similar to the results of Sullivan et al. (22). Our measurements of PA also showed that Raw(f) was essentially constant over the entire f range, thereby demonstrating that the low-frequency variation in RL( f ) was due to the rheological properties of lung tissue, as has been shown in normal
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
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#3 a I I
i
0
2
4
6
8
10
f (Hz)
5. Rti( f) vs. f from each of the 2 alveolar pressures measured in 3 under control conditions (solid line) and with 16 (dash-dot line) and 64 mg/ml MCh (dashed line). FIG. rabbit
dogs (10). In addition, we found that Rti( f ) became close to zero above 4 Hz, similar to dogs (3). This has practical significance for the study of airway phenomena in rabbits, because it means that Raw(f) is given by RL( f) at f > 4 Hz. In contrast, at lower f, RL( f ) reflects a variable degree of Rti( f), comprising as much as 88% of the former at 0.16 Hz. We note also that our results apply to both the older group of rabbits ( 1-3) and the younger group (4-6). 0 f course, with only three animals in each group, we cannot draw strong conclusion .s about the efNevertheless the only difference befects of maturity tween the younger and older rabbits apparent from Fig. 2 is that the values of RL( f) are considerably higher in the younger rabbits as a consequence of their smaller sizes. The values of RL(mv) were somewhat larger than the corresponding values of RL( l), RL,, being on average 130% of Rx,(l) (Table 1). These differences are relatively small compared with the variation in RL( f) occurring between 0.16 and 4 Hz (Figs. 1 and 3) and probably reflect some nonlinear aspect of the respiratory system. Our results indicate that the most important nonlinearity is the flow dependence of Raw, because Raw(l) was smaller than Raw(mv), whereas there was no difference between Rti( 1) and Rti(mv) (Table 1). One might have expected there also to have been differences between RL( 1) and RL(mv) because of the differences in mean
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lung vol .ume betwee n the forced oscillation and tidal ventilation tech .niques. However, this should have resulted in differences between Rti( 1) and Rti(mv), because tissue viscance has been reported to be volume dependent (15). Presumably the conditions of our experiment were such that volume-dependent effects were small, in contrast, for example, to the study of Lutchen and Jackson (17), who found a tidal volume dependence of the tissues when oscillating around functional residual capacity in dogs. The progressive heterogen .eity of alveolar ventilation th .at became apparent in the rabbits exposed to increasing doses of MCh (see RESULTS and Fii. 5) is similar to that reported in dogs after inhalation of histamine (16) and presumably refl .ected a combination of inhomogeneous aerosol deposition and inhomogeneous responsiveness throughout was the lun g. This inhomogeneity also presumably resp ionsible for the occurrence of physically- meaningless negative values of Rti( f ). Indeed, Fredberg et al. (7) also found negative Rti values after histamine exposure in dogs, which they suggested was due to an airway shunt compliance. Their theory might also explain our own results. Alternatively, our negative Rti may have resulted from parallel inhomogeneities across the lung. That is, we used the V measured at the trachea to calculate regional Rti in an inhomogeneous lung, whereas in reality the V entering each alveolar region was not proportional to tracheal V. Regional V must be a function of the mechanical properties of the region concerned compared with those of other regions that compete for the total V. In any case, compartmentalization of thea lung in either a parallel or serial 1fashion means that V at the trachea does not reflect V at the alveolar level, which can potentially result in the calculation of a negative Rti. We also found variable relative responses to MCh at the low and high ends of the frequency-spectrum studied, as can be discerned in Fig. 2. The ratio of R~(0.16) to R~(10.64) had an average value of 6.08 t 3.21 under control conditions. At the highest doses of MCh represented in Fig. 2, this ratio had changed by factors of 0.58, 1.63, 0.43, 2.18, 1.18, and 1.20 for rabbits 1, 2, 3, 4, 5, and 6, respectively. Thus some rabbits experienced a greater relative increase in RL( f) at low frequencies, reflecting predominantly tissue viscoelastic properties, whereas others had a greater change at high frequencies where the resistance of the airways is of much greater importance. Such variability with MCh has been noted elsewhere in dogs (17) and rabbits (19). The three less mature rabbits developed lobar atelectasis during the MCh c hallenge. Interestingly, this suggests that age may be an importan t factor in the response of rabbits to MCh. This is consistent with in vitro-findings of greater airway smooth muscle contractility in immature rabbits (13, 23) and pigs (20) than in their mature counterparts. Lobar atelectasis during bronchial challenge has also not been reported in mature dogs instrumented with alveolar capsules. Although the occurrence of lobar atelectasis obliterated our PA signals and so prevented assessment of regional tissue properties at high MCh concentrations, the RL( f) measurements continued to indicate overall changes in lung mechanics in an expected manner (Figs. 2-4). For example, we noticed that the occurrence of atel-
Downloaded from www.physiology.org/journal/jappl at Tulane University (129.081.226.078) on February 15, 2019.
LOW-FREQUENCY
IMPEDANCE
ectasis in the three younger rabbits (4-6) tended to be accompanied by a particularly large increase in RL( f) at the higher f (Fig. 4). We suspect that this can be explained in part by the fact that atelectasis suddenly reduces the total volume of the lung and consequently increases its resistance simply due to loss of available airways. An increase in elastance due to loss of available lung volume should accompany this atelectasis, and indeed we noticed that EL(~) below 1.36 Hz (Fig. 3) also shows the same tendency to increase more markedly with the occurrence of atelectasis. Finally, although the data obtained in the present study pertain to the lung alone, we can speculate on how our results might carry over to the intact rabbit in which tracheal pressure-flow relationships are also influenced by the chest wall. The tissues of the chest wall are highly viscoelastic and so contribute significantly to respiratory impedance at low frequencies (1, 3). Also, the chest wall has been shown in dogs to have a component that behaves similarly to Raw, as determined by the flow interruption technique (2, 3), which would contribute to respiratory impedance at frequencies > 5 Hz, where viscoelastic dissipation of energy no longer occurs. It is quite likely, then, that total respiratory impedance in rabbits would exhibit noticeable differences compared with ZL alone. However, given that bronchoconstriction presumably affects the lung exclusively, we would expect that the differences between respiratory impedance and ZL would become relatively less important as bronchoconstriction proceeds. For the same reason, changes in lung mechanics could be accurately inferred from changes in total respiratory mechanics. In summary, we have found that the forced oscillation technique provides a convenient and informative way of characterizing pulmonary mechanics in rabbits under control conditions and during severe bronchoconstriction. Furthermore the ZL and Zti spectra obtained show that the mechanical properties of the rabbit lung are qualitatively similar to those of other species. In particular, we found that the f range we used is useful for assessing pulmonary mechanics, because it provides information pertaining to the lung tissues at f < 4 Hz while, at least under control conditions, reflecting exclusively airway properties above 4 Hz. Although we cannot be sure that< the same is true during bronchoconstriction because of the lung inhomogeneities induced, if Rti( f) also falls to zero above 4 Hz with MCh, then RL( f) above this fcontinues to be a measure of overall Raw. This work was supported by the Medical Research Council of Canada (MRC) and the EL/JTC Memorial Research Fund. R. Tepper was supported by The American Lung Association of Indiana; J. G. Martin is a Scientist of the MRC; J. H. T. Bates was a Scholar of the MRC; and J. Sato was supported by Chiba University Hospital, Chiba, Japan. Address for reprint requests: J. H. T. Bates, Meakins-Christie Laboratories, 3626 St. Urbain St., Montreal, Quebec H2X 2P2, Canada. Received 18 January 1991; accepted in final form 27 February 1992. REFERENCES 1. BARNAS, G. M., K. YOSHINO, S. H. LORING, AND J. MEAD. Impedance and relative displacements of relaxed chest wall up to 4 Hz. J. Appl. Physiol.
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