INTRODUCTION

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SMALL RODENTS ARE NOW WIDELY utilized as animal models of lung disease because they can be obtained easily in large numbers for a reasonable price, they grow rapidly, and pure-bred strains are available. Ultimately, of course, to apply results from such studies to human disease, we need to know how rodent lungs compare mechanically with human lungs. However, it is also important to understand how the respiratory mechanical properties of different rodents compare among species so that appropriate choices can be made as to which species to use when a particular disease state is modeled. A number of studies have attempted to characterize and compare respiratory mechanics in different mammalian species (4, 10, 21, 27), but most of them consisted of pooled data from different sources in the literature, and some even used recently killed animals. Furthermore, these comparative studies have not taken into account the well-known dependence of respiratory mechanics on frequency and volume. We thus identified a need for a comprehensive, comparative study of respiratory system impedance (Zrs) in a variety of small-animal species that are currently widely used in respiratory research.

Particular interest has been directed recently to the evaluation of the mechanisms that affect respiratory system properties under both healthy and abnormal conditions and whether the predominance of these effects is in the airways or the parenchymal and/or chest wall tissues (1, 3, 12, 15-17, 19, 25-31, 35). Respiratory system elastance is known to increase with frequency, and tissue resistance is known to decay hyperbolically over the range of physiological breathing frequencies, whereas airway resistance (Raw) remains fairly constant both in healthy and mildly constricted lungs (12-15, 22, 32, 37). Consequently, the relative contributions of airway and tissue resistances are highly dependent on the breathing frequency. Furthermore, these relative contributions can change with lung volume (17). This highlights the importance of measuring Zrs over a broad range of frequencies and volumes. Such an undertaking is not trivial because of the technical difficulties associated with measuring Zrs accurately in small animals. However, we now have at our disposal a computer-controlled, small-animal ventilator (SAV) (34) that has solved these difficulties and allows us to measure Zrs over a wide frequency range and at controlled lung inflation pressures in animals as small as a mouse.

The goal of the present study was, therefore, to make a comparative study of Zrs in normal mice, rats, guinea pigs, and rabbits over a broad range of frequencies and at different levels of lung inflation with the use of the SAV. We interpreted the measured Zrs in terms of a model proposed by Hantos et al. (15), which features a Newtonian resistance (R) connected to a constant-phase viscoelastic tissue compartment. The parameters of the model account extremely well for Zrs <20 Hz and permit us to compare interspecies respiratory mechanics in a frequency-independent fashion.

	MATERIALS AND METHODS

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Experimental procedures. We studied four different species of rodents: A/J mice (n = 11; 20.5-24.7 g), Sprague-Dawley rats (n = 8; 260-290 g), Dunkin-Hartley guinea pigs (n = 5; 360-410 g), and New Zealand White rabbits (n = 6; 2.6-3.0 kg). Mice and rats were sedated with an intraperitoneal bolus of xylazine (7 and 14 mg/kg, respectively). All animals were anesthetized [pentobarbital sodium in mice (50 mg/kg ip), rats (35 mg/kg ip), and guinea pigs (65 mg/kg ip); sodium thiopental in rabbits (35 mg/kg iv)] and tracheostomized. Except for the rabbits, which had their trachea cannulated with a rigid plastic tube and which were ventilated with a commercial version of the SAV (flexiVent, SCIREQ, Scientific Respiratory Equipment, Montreal, Quebec), all of the other animals had a snug-fitting metal tracheal cannula connected to a SAV prototype (34). Mice, rats, guinea pigs, and rabbits were mechanically ventilated with sinusoidal inspiration and passive expiration with tidal volumes of 6, 8, 9, and 7 ml/kg and breathing frequencies of 150, 90, 70, and 60 breaths/min, respectively. Positive end-expiratory pressure (PEEP) was set at 3 hPa by connecting the expiratory line of the ventilator to a water trap. Mice and rabbits were paralyzed with pancuronium bromide (0.8 mg/kg ip and 0.1 mg/kg iv, respectively). Rats and guinea pigs were paralyzed with succinylcholine chloride (20 and 13 mg/kg ip, respectively).

Data analysis. Corrections for gas compressibility within the system and resistive and accelerative losses in the connecting tubing and tracheal cannula were performed as described previously (3, 17). Briefly, we first obtained dynamic calibration signals of Pcyl and Vcyl from the SAV before connecting the animal by applying the volume perturbation through the tracheal cannula first when it was completely closed and then again when it was open to the atmosphere. Subsequently, when the animal was connected to the SAV, we used these calibration signals to remove the contribution of both the tracheal cannula and the SAV itself from the measured animal Zrs.

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	RESULTS

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Figures 1 and 2 show, respectively, NRrs and NXrs as functions of frequency for each of the species studied at five different levels of PEEP. In each case, NRrs decreased monotonically, whereas NXrs increased monotonically with frequency. The resonant frequency was not attained in any of the animals by 9.125 Hz, as NXrs was always negative up to this frequency. Figure 1 also shows that mice had lower NRrs values at higher frequencies than did rats, guinea pigs, or rabbits. At the lowest frequency of 0.25 Hz and at 0-hPa PEEP, mice and rabbits had higher NRrs than did rats and guinea pigs. Above 6 Hz, NRrs decreased consistently with increasing PEEP. Figure 2 shows that mice and rabbits had lower NXrs at 0.25 Hz, in the absence of PEEP, than did rats and guinea pigs.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Real parts of respiratory system impedance normalized to body weight (BW) (NRrs) in 4 different species. Values are means ± SE calculated for positive end-expiratory pressures (PEEPs) ranging between 0 and 7 hPa in each species.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Imaginary parts of respiratory system impedance normalized to BW (NXrs) in 4 different species. Values are means ± SE calculated for PEEPs ranging between 0 and 7 hPa in each species.

Figure 3 shows the PEEP dependence of the Zrs model parameters in the four species studied. NR tended to decrease with PEEP in all species and was very similar in rats and guinea pigs, lowest in mice, and highest in rabbits. NI was very small in magnitude and usually negative. Furthermore, when I was excluded from Eq. 1 and the model was refitted to the Zrs data, there was essentially no change in the values of the remaining parameters. We, therefore, consider the influence of NI to be negligible over the frequency range studied. Mice and rats had higher values of NGti in the absence of PEEP than when PEEP was applied. NGti did not show any PEEP dependence in rabbits, and in guinea pigs NGti tended to increase at higher levels of PEEP. Differences in NGti among species were minor and occurred at a PEEP of 0 hPa between mice and rats and at PEEPs of 2 and 7 hPa between rats and rabbits. In all species, NHti tended to decrease with increasing PEEP until 5 hPa when there was an inflexion point, except in mice. At lower PEEPs, mice and rabbits had higher values of NHti than did rats and guinea pigs. The  and  were independent of PEEP in mice, but they showed a tendency to decrease and increase, respectively, with PEEP in rats and rabbits. In guinea pigs, the only difference noted was between PEEP levels of 0 and 2 hPa. Except when no PEEP was applied, mice showed values of  and  that were significantly different from those of other species.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Parameters of the constant-phase model fitted to respiratory system impedance data of mice (n = 11), rats (n = 8), guinea pigs (n = 5), and rabbits (n = 6) as a function of PEEP. Values are normalized to BW and represent means ± SE. NR, normalized Newtonian resistance; NI, normalized inertance; NGti, normalized tissue damping; NHti, normalized tissue elastance;  = (2/)arctan(Hti/Gti); , hysteresivity.

The repeat set of measurements that was made in each animal was analyzed to establish the reproducibility of the results in time. The first and second measurements of R, Gti, Hti, , and  at each of the five different levels of PEEP were compared. Paired t-test analysis showed no significant differences in almost all the parameters. Exceptions were found for Gti in guinea pigs at a PEEP of 7 hPa, for Hti in guinea pigs at PEEPs of 5 and 7 hPa, for Hti in rabbits at a PEEP of 0 hPa, and for  and  in mice at a PEEP of 2 hPa. However, the relative differences were always <9.1% for Hti and , <3.1% for Gti, and <1% for .

	DISCUSSION

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The present study was motivated by the wide utilization of rodents in the investigation of lung development and processes related to respiratory-system diseases. Although the species we used have been studied in the past (3, 4, 10, 14, 16, 17, 19, 21, 25-30, 32, 35-37, 39), very little has been reported about the Zrs of mice, no doubt in part because of the technical difficulties associated with making the necessary measurements. Also, most of the comparative studies on mammalian respiratory mechanics have been performed during spontaneous tidal breathing, which allows animals to choose (and vary) their tidal volumes, breathing frequencies, and functional residual capacities. Furthermore, existing comparative data have been gathered from different sources in the literature; therefore, the methods used to assess mechanics were not always the same in the various species. Considering the present need to perform accurate measurements of Zrs in small rodents and the fact that Zrs depends markedly on frequency and lung volume (Figs. 1 and 2), there is clearly a need for comparative measurements of Zrs in various rodent species over a broad range of frequencies and at different levels of PEEP. This was the purpose of our study.

Zrs by itself can provide useful information regarding the state of the respiratory system, but the utilization of suitable models can greatly facilitate its interpretation. The model we used consists of an R and I leading to a constant-phase tissue compartment characterized by the dissipative parameter Gti and the elastic parameter Hti. It has been shown previously that this model provides extremely accurate fits to normal Zrs over the range of frequencies that we studied (12, 15, 17, 22, 31), despite having only four independent parameters. As in our laboratory's previous study in rats (17), we found I to make a negligible contribution to input impedance over the frequency range examined in the present study. Our laboratory's previous studies in dogs (2) and rats (17) have also shown that R contains contributions from both the chest wall tissues and the pulmonary airways, respectively, to varying degrees, depending on the species. Gti and Hti characterize the viscoelastic properties of the respiratory tissues.

To compare Zrs, R, I, Gti, and Hti among species, we multiplied them by BW. Alternatively, we could have normalized to lung volume. However, this can be misleading because lung volume at any particular inflation pressure depends on the elastic properties of the lungs and chest wall (23), and because respiratory elastance (Hti) is one of the parameters in which we are interested, we would be effectively normalizing it to itself. On the other hand, we could have normalized to lung weight. However, it is known that guinea pigs undergo marked bronchoconstriction after death, probably due to the release of substance P, which can increase vascular permeability and enhance edema formation (20). Therefore, the postmortem measurement of lung weight in guinea pigs would likely overestimate its value in vivo relative to the other species. Consequently, we decided that normalizing to BW was the most appropriate thing to do, especially as our mechanics parameters reflect not only the lungs but also chest wall properties (8). Also, the lungs of an animal serve its entire body and not just its lung tissue; therefore, it would seem to make sense to consider how our parameters relate to the entire body. Finally, normalizing a parameter with respect to BW allows it to be easily associated with metabolic rate, which has been shown to follow BW to the 0.75 power in mammals (33).

NR and Raw. Our results show that NR tends to decrease with PEEP (Fig. 3), presumably due to increasing parenchymal tethering forces causing an increase in airway caliber. Moreover, NR increased from the smaller to the larger species. This appears to suggest that the airways of smaller species are relatively larger than those of larger species, although we must be careful in drawing such a conclusion because R contains contributions from both the airways and the chest wall tissues, although the relative amounts may vary with species. In dogs, for example, the chest wall and airways have been shown to make approximately equal contributions to the Newtonian resistive properties of the respiratory system (2), whereas in humans the chest wall has been reported to contribute 27% of R (5). Data collected by Rotger et al. (32) suggest that the chest wall contributes roughly one-third of R in rabbits. We have observed in mice that the lung contributes up to 50% of R over a range of lung volumes (unpublished observations), whereas in rats (17, 30) and cats (12) the chest wall contributes very little to the Newtonian properties of the respiratory system. Therefore, it is possible that the rank ordering of NR that we found in the present study (Fig. 3) may not be the same rank ordering of actual normalized Raw.

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View larger version (18K): [in this window] [in a new window]   Fig. 4.   Allometric relationships of the respiratory system model parameters obtained in normal mice (n = 11), rats (n = 8), guinea pigs (n = 5), and rabbits (n = 6) at 0-hPa PEEP. R, Newtonian resistance; Gti, tissue damping; Hti, tissue elastance. Solid line corresponds to the linear regression analysis of each parameter with BW on a log-log scale. r2, Coefficient of determination; a and b, variables.

Resonant frequency. The resonance frequency was not reached by 9.125 Hz in any of the species we investigated (Fig. 2). This agrees with data obtained from studies on low-frequency forced oscillations in rats (14, 17), guinea pigs (39), and rabbits (37) and from studies on high-frequency oscillatory ventilation in rabbits (36). Petak et al. (30) were able to detect an inertive component in the respiratory system properties of rats using frequencies up to 21 Hz and concluded that the inertive component of Zrs is essentially determined by lung impedance, agreeing with data obtained from cats (12). The fact that our estimates of I were negligible for frequencies <9.125 Hz (Fig. 3) does not mean that there were no inertial elements in the airways but rather that inertial effects were minimal over the frequency range studied.

Effect of PEEP on tissue resistance. We found NGti in mice and rats to be higher at lower levels of PEEP (Fig. 3), agreeing with previous finding in rats (17). It has been shown in both rats and dogs that the chest wall resistance is independent of mean Pao (1, 17). Therefore, the negative dependence of Gti with lung volume is fully attributable to the lung and most likely reflects air space closure occurring at lower levels of PEEP. However, in rabbits, NGti was not dependent on end-expiratory volume, and in guinea pigs, there was a slight rise in NGti at the highest level of PEEP compared with intermediate lung volumes (Fig. 3). Because smaller animals are known to have more compliant chest walls, the relaxation volume normalized to lung size in these animals is usually lower than in larger animals (10). Therefore, the mice and rats in this study could have been ventilated at proportionately lower lung volumes at a given level of PEEP compared with other rodents, thus explaining the differences we found among species.

Effects of PEEP on Hti. The dependence of respiratory system elastance on PEEP was marked by a decline in NHti at lower levels of lung inflation in all species followed by an increase at 5 hPa, except in mice (Fig. 3). These results confirm a previous study in rats (17), in which Hti as a function of lung volume for both the lung and chest wall exhibited minima. Presumably the increasing values of NHti at low levels of PEEP reflect alveolar collapse and/or airway closure resulting from reduced airway-parenchymal interdependence forces, although nonlinear chest wall elastic properties may also have contributed to this phenomenon. The increase in NHti at higher levels of lung inflation was probably due to the nonlinear elastic tissue properties of both chest wall and lungs. The absence of a clear minimum in the NHti curve for mice may reflect the fact that smaller animals with comparatively floppier chest walls have relatively lower lung volumes at a given PEEP. The range of PEEPs studied would then not have been sufficient to push the lungs and chest wall into the upper nonlinear portions of their respective pressure-volume curves. Another possibility is that an increase in NHti for the lung may have been compensated for by a decrease in NHti for the chest wall, because we know that in rats the minima in these two quantities occur at different inflation volumes (17). The lung volume dependence of pulmonary elastance has been previously studied in small rodents with the use of the alveolar capsule technique (19, 25, 26, 28, 29, 35), and in all animals a sharp increase in dynamic elastance with lung inflation has been reported. Nagase et al. (25, 26, 28, 29) studied lung mechanics at transpulmonary pressures as low as 3 hPa in mice, rats, guinea pigs, and rabbits but could not detect a fall in elastance at very low lung volumes, confirming the results of Shardonofsky et al. (35) in rabbits ventilated against PEEPs of 2-4.6 hPa. Most probably, the difference between our results and theirs is due to the fact that we used very-small-amplitude volume oscillations, whereas the alveolar capsule technique was performed during tidal ventilation.

Tissue mechanics among species. Leith (21) showed that chest wall compliance increases with BW to the 0.86 power, whereas lung compliance increases with BW to a power between 1.08 and 1.20. When Leith used respiratory system elastance in the equivalent of Eq. 4, he found a value for b of 1.04, which is the exact value we found for Hti (Fig. 4). However, mice and rabbits had relatively more rigid respiratory systems at low PEEPs than did rats and guinea pigs (Fig. 3). This may have been due to differences in the relative compliances of the lung and chest wall among species. However, the chest wall contribution to respiratory system elastance is ~35% in mice (16) and 20% in rabbits (32). Therefore, it seems unlikely that a relatively increased chest wall elastance in rabbits would solely account for a higher NHti in rabbits than in rats or guinea pigs.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Elastances normalized to BW and relative to the mouse values as a function of BW in different species. NE,L, normalized pulmonary elastance estimated from total alveolar volume obtained from Mercer et al. (24); NHti,L, normalized pulmonary tissue elastance.