Variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry

J. Jane Pillow,1,2 Alan H. Jobe,3 Rachel A. Collins,1 Zoltán Hantos,1,4 Machiko Ikegami,3 Timothy J. M. Moss,5 John P. Newnham,2,5 Karen E. Willet,1 and Peter D. Sly1,2

1Centre for Child Health Research, 5School of Women's and Infants' Health, The University of Western Australia, and 2King Edward and Princess Margaret Hospitals, Perth 6008, Australia; 3Cincinnati Children's Hospital Medical Center and University of Cincinnati School of Medicine, Cincinnati, Ohio 45229-3039; and 4Department of Medical Informatics and Engineering, University of Szeged, H-6720 Szeged, Hungary

Submitted 4 May 2004 ; accepted in final form 5 July 2004


    ABSTRACT
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 DISCUSSION
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Antenatal exposure to intra-amniotic (IA) endotoxin initiates a complex series of events, including an inflammatory cascade, increased surfactant production, and alterations to lung structure. Using the low frequency forced oscillation technique as a sensitive tool for measurement of respiratory impedance, we aimed to determine which factors contributed most to measured changes in lung mechanics. Respiratory impedance data obtained from sedated preterm lambs exposed to either IA injection with saline or 20 mg of endotoxin 1, 2, 4, and 15 days before delivery at 125 days gestation were studied, and association with indexes of standard lung morphometry, inflammatory response, and alveolar surfactant-saturated phosphatidylcholine (Sat PC) pool size was demonstrated. Reduction in tissue impedance with increasing interval between exposure and delivery was evident as early as 4 days after IA endotoxin injection, coinciding with resolution of inflammatory reaction, increased alveolar surfactant pools, and contribution of alveolar ducts to the parenchymal fraction, and a later decrease in the tissue component of the parenchymal fraction. Decreases in tissue damping (resistance) were more marked than decreases in tissue elastance. Log alveolar Sat PC accounted for most variability in tissue damping (88.9%) and tissue elastance (73.4%), whereas tissue fraction contributed 2 and 6.4%, respectively. The alveolar Sat PC pool size was the sole factor contributing to change in tissue hysteresivity. No changes were observed in airway resistance. Despite the complex cascade of events initiated by antenatal endotoxin exposure, variability in lung tissue mechanics is associated primarily with changes in alveolar Sat PC pool and lung morphology.

fetal organ maturity; lung compliance; oscillometry; respiratory mechanics


EARLY INSULTS TO THE LUNG may have a significant impact on its long-term structural and functional integrity. Although animal models have advanced our understanding of these pathways, there is increasing interest in using sensitive physiological tools to follow changes in the structure-function relationships within the lung and to assist evaluation of treatments aimed to prevent long-term sequelae of lung disease. Understanding the impact of known confounding factors to the variability of physiological measurements in established models of respiratory disease is an important step toward application of these tools.

Antenatal inflammation following infection has been increasingly recognized as a forerunner to the development of chronic lung disease in the newborn infant, which may result even in the absence of respiratory disease immediately after birth (12). We have established a preterm sheep model of inflammation in the newborn lung using intra-amniotic (IA) endotoxin (5). Jobe et al. (4) reported the impact of the exposure time between an antenatal IA endotoxin injection and subsequent delivery at 125 days gestation on lung function, finding evidence of proinflammatory cytokines within 5 h of exposure, inflammation by the end of the first day, and increased surfactant pool sizes by day 2. Despite such prompt responses to endotoxin exposure, functional improvements in oxygenation, ventilatory efficiency index, lung compliance, and lung volumes at 40 cmH2O were not evident on ANOVA testing until 7 days after exposure. Changes in lung morphometry are evident 6 days after IA endotoxin injection (13); however, the duration of exposure required to induce these changes has not been reported, and, consequently, the interrelationship between structural and functional changes under these conditions is not known.

A barrier to ascertaining the interrelationship among cellular, mechanical, and structural changes has been the relative insensitivity of routine respiratory mechanics measurements to identify the functional changes that result from effects occurring early in the inflammatory cascade, such as increased surfactant pool sizes 2 days after IA endotoxin. The low-frequency forced oscillation technique (LFOT) is a sensitive and minimally invasive measure of airway and tissue mechanics that detects changes in preterm lamb lung function sooner than can be achieved with other lung function techniques (8). During forced oscillations, a pressure signal composed of a range of frequencies is applied at the airway opening. The relationship between pressure and flow at each of the component frequencies is determined and plotted, establishing the impedance spectra. Variables representing the mechanical properties of the airways (resistance and inertance) and the tissues (resistance or "damping" and the elastance) can be obtained by fitting a model to the impedance spectra (3).

Low-frequency forced oscillatory mechanics measurements were obtained in a subset of animals exposed to IA endotoxin injection at different time intervals before delivery at 125 days gestation (4). Our main goal was to gain insight into structure-function relationships in the lung and ascertain the likely mechanisms primarily responsible for these changes by studying associations between these more detailed and specific lung function data and previously reported indexes of inflammation, surfactant pool size, and previously unreported lung morphometry.


    METHODS
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 METHODS
 RESULTS
 DISCUSSION
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Ethics

Studies were undertaken on 35 preterm lambs born via cesarean section at 125 days gestation to date-mated Merino ewes. The investigations were approved by the animal ethics committee of the Western Australia Department of Agriculture and Cincinnati Children's Hospital Medical Center.

Animals, Antenatal Endotoxin Treatment, Delivery, and Postnatal Care

The antenatal preparation and postnatal care of animals in this study have been extensively documented previously (4, 5). Briefly, date-mated Merino ewes received one 20-mg dose of Escherichia coli 055:B5 endotoxin (Sigma, St. Louis, MO) by ultrasonically guided IA injection at either 1, 2, 4, or 15 days before delivery at 125 days gestation by cesarean section (4). Control ewes received saline instead of endotoxin. The sedated singleton fetus was ventilated for 40 min after delivery with a time-cycled, pressure-limited infant ventilator (4). Peak inspiratory pressure was adjusted to maintain tidal volume (VT) <10 ml/kg and was not increased beyond 40 cmH2O. Lower respiratory system impedance (Zlrs) was measured at the prevailing mean airway pressure at 35 min postnatal age in a subset of these animals. Lambs were killed at 40 min with 30 mg/kg of pentobarbitone, the tracheal tube was clamped, and the lungs were excised. Investigators treating the newborn animals and those undertaking physiological, morphometric, and laboratory investigations were blinded to treatment group assignment.

Forced Oscillatory Mechanics

Zlrs was obtained using the LFOT as previously described (8) except that transrespiratory pressure was measured in the distal trachea, and measurements were obtained at the prevailing mean airway pressure (dependent on the ventilator settings at time of measurement). The lung volume during oscillatory measurements was estimated by linear regression over relevant portions of the deflation pressure-volume curves obtained from the excised lungs (4) and interpolation of the prevailing mean airway pressure applied during the measurement. Zlrs spectra computed from four paired measurements of tracheal pressure and oscillatory flow at each time point were averaged, and a model comprising an airway compartment with a frequency-independent (Newtonian) resistance (Raw), an inertance (Iaw), and a constant-phase tissue compartment characterized by coefficients of tissue damping (resistance; G) and elastance (H) was fitted to the averaged spectra (3). Tissue hysteresivity ({eta}) represents the interdependence of tissue damping and tissue elastance and was calculated as the ratio of G and H (2).

Morphometry

All morphometric assessments were performed by a blinded observer (K. E. Willet or R. A. Collins) on the right upper lobe (RUL), which was fixed at 30 cmH2O via bronchial instillation of 4% phosphate-buffered paraformaldehyde. Fixed lobe volume was measured by volume displacement (10). Lobes were cut into 5-µm serial sections, and three hematoxylin and eosin-stained sections were selected randomly for morphometric evaluation (1). A cycloid grid was used to estimate pleural, perilobular connective tissue, nonparenchymal, and presumptive aerated and nonaerated parenchymal fractions (13). The total volume of each compartment was derived from volume fraction multiplied by fixed lobe volume.

Digitized images captured from 10 nonoverlapping parenchymal fields in each section were examined at x950 magnification (13). The relative size and the presence of secondary alveolar septa distinguished alveolar ducts from alveoli, whereas size and morphology were used to identify alveoli distinct from saccules. The number of points that fell on air space alveolar septal tissue and the number of air/tissue-tissue/air intercepts were quantified to determine the alveolar, ductal, and alveolar wall volume and area indexes.

Surfactant Pool Size, Alveolar Inflammatory Cell Counts, and Lung Volume

The effect of delay between IA endotoxin injection and delivery on inflammation scores within the air space and tissue compartments and the surfactant pool size from the left lung were reported previously (4). Only those data pertaining to the lambs included in the current study were combined with morphometric data from the same lungs for the interpretation of changes in tissue mechanics.

Statistical Analyses

Changes in lung function and morphometric parameters at each different interval between the IA injection (endotoxin or saline) and birth were assessed using one-way ANOVA with post hoc analysis of differences between the control and treatment groups using Dunnett's two-sided t-tests. Significant associations between indexes of oscillatory lung mechanics and inflammatory cell counts, surfactant pool size, and morphometric indexes were initially ascertained from bivariate correlations using Spearman's rho. Where significant associations were present and a nonlinear relationship existed, data were transformed to linearize the relationship. Pearson's correlation coefficient was then determined for each significant association. The contribution of factors with significant associations to the variability observed for each lung function parameter was determined using stepwise multiple linear regression indexes. Factors were entered into the model in order of the strength of their Pearson correlation coefficients and retained in the regression model if their inclusion resulted in a significant (P < 0.05) step change in the r2 value.


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Postnatal Characteristics and Outcome

Table 1 lists a summary of the animal characteristics, lung volumes, surfactant production, and indexes of lung morphometry and inflammation for the subset of animals from each treatment group in which both forced oscillatory mechanics and morphometry measurements were obtained. The postnatal clinical outcome parameters of each of the subgroups included for these measurements were reported previously in a larger group of animals (4).


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Table 1. Animal characteristics and indexes of lung surfactant, inflammation, and morphometry

 
Morphometry

Representative histological sections from each of the study groups are shown in Fig. 1. There was no difference in total fixed volume of the RUL, although there was an increase in total parenchymal volume (P = 0.03) alongside a decrease in total nonparenchymal volume (P < 0.001; Table 1), resulting primarily from a decrease in the perilobular connective tissue 24 h after IA endotoxin injection.



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Fig. 1. Representative tissue morphology after intra-amniotic (IA) endotoxin exposure. Representative hematoxylin and eosin-stained sections of lung tissue obtained in 125-day lambs after varying lengths of exposure to either IA saline or endotoxin before delivery. A: saline (control); B: 1 day; C: 2 days; D: 4 days; E: 15 days.

 
There was a noticeable change in the composition of the parenchymal compartment in response to increasing interval between IA injection of endotoxin or saline and delivery, with an increase in the ductal air space at the expense of alveolar air space and a trend toward a decrease in alveolar volume (P = 0.06) (Table 1). In addition, a decrease in the tissue fraction was evident in the 15-day group. Alveolar numerical density tended to be lower than the control group after 2 days of IA endotoxin injection, but there was no significant change in the total number of alveoli in the RUL within the limited group of animals reported in this study. The surface area of alveoli and alveolar ducts decreased as a percentage of parenchymal volume, although total surface area was unchanged when related to fixed lobe volume. The lung volume during forced oscillatory mechanics increased with increasing delay between IA endotoxin injection and delivery (Table 1).

Forced Oscillatory Mechanics

Impedance spectra. Representative impedance spectra for the control animals and lambs studied 15 days post-IA endotoxin injection are shown in Fig. 2. IA endotoxin injection resulted in a marked decrease in resistance values and an elevated reactance corresponding to more compliant lungs.



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Fig. 2. Representative impedance spectra are shown for a control lamb ({circ}) and 1 exposed to endotoxin 15 days before delivery. A: resistance of the lower respiratory system (Rlrs) at each of the component frequencies of the forcing function representing the components of impedance in phase with flow. B: reactance of the lower respiratory system (Xlrs) representing the components of impedance out of phase with flow (elastance and inertance).

 
Parameter estimation. Forced oscillatory measurements were technically invalid in two of the seven control animals studied, owing to computer malfunction. IA endotoxin injection decreased both G (P < 0.001, Fig. 3A) and H (P = 0.002, Fig. 3B), with progressively greater reductions seen with increasing interval between endotoxin injection and delivery. Multiple comparison post hoc testing showed a significant effect at 15 days postexposure for both G (P = 0.003) and H (P = 0.024), and a strong trend toward a decrease in the 4-day group for G (P = 0.054) was also evident. The earlier effect of IA endotoxin on G compared with H was evident as a decrease in {eta} (P < 0.001, Fig. 3C) that was highly significant at both 4 days (P = 0.006) and 15 days (P < 0.001), with a strong trend observed at 2 days following exposure (P = 0.07) compared with control animals. There was no significant effect of IA endotoxin on Raw (P = 0.21, Fig. 3D), whereas respiratory compliance determined from VT and the ventilatory pressure settings only showed a significant effect at 15 days (P = 0.001). Respiratory reactance remained negative in the measured frequency range, reflecting the dominance of the elastic properties of the respiratory system and the small influence of the inertive properties. Consequently, the values of Iaw were weakly estimated by the model fitting, and they are not reported.



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Fig. 3. Influence of IA endotoxin on lung mechanics. Mechanical parameters of the lung in lambs exposed to endotoxin at varying intervals [1, 2, 4, and 15 days (d)] before delivery at 125 days gestation obtained (A–D) from forced oscillatory mechanics and from dynamic compliance (Cdyn; E) at the ventilatory frequency. P values indicate significance of changes on one-way ANOVA after accounting for treatment group. Dunnett's t-test was used for post hoc analysis of within-group changes vs. the control group (C). G, coefficient of tissue damping (resistance); H, coefficient of tissue elastance; {eta}, hysteresivity (G/H); Raw, airway resistance. *P < 0.05, #P = 0.07.

 
Correlations and Multiple Linear Regression

The significant associations between the tissue mechanics and morphometric, inflammatory, surfactant, and volume data in which effects of treatment group assignment were evident are summarized in Table 2. Stepwise multiple linear regression analysis identified the log alveolar surfactant pool during oscillatory mechanics to be the single most significant factor contributing to the variability in G (88.9%), H (73.4%), and {eta} (80.4%) (see Fig. 4). In addition, the tissue fraction contributed a further 2% to the variability associated with G and 6.4% to the variability in H.


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Table 2. Bivariate Pearson's correlations between lung mechanics, surfactant pool size, inflammatory, and morphometric indexes

 


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Fig. 4. Correlation of tissue mechanics with log alveolar saturated phosphatidylcholine (Sat PC) pool size. Data highlighting the strong correlation between indexes of tissue mechanics and the size of the alveolar Sat PC pool in lambs after IA exposure to endotoxin at varying intervals (1, 2, 4, and 15 days) before delivery at 125 days gestation. A logarithmic scaling is used on both axes. The regression lines were derived from linear regression on the logarithmic data. {circ}, Control (saline); {square}, 1-day exposure; {triangleup}, 2-day exposure; {lozenge}, 4-day exposure; hexagon, 15-day exposure.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Using a previously reported model of antenatal endotoxin exposure (4), we combined published data on the inflammatory and surfactant response with new information detailing the changes in lung morphometry and partitioned measurements of airway and tissue mechanics after differing durations of in utero endotoxin exposure in a subset of the original study group. The pooled data were used to advance our understanding of the interrelationship between structure and function of the newborn lung and the interpretation of minimally invasive measurements of forced oscillatory mechanics. There was a complex pattern of changes in morphometric indexes with increasing interval between IA endotoxin injection and delivery, including a decrease in nonparenchymal tissue fractions and a change in the balance between alveolar and ductal contributions to parenchymal air space and later thinning of alveolar walls. Using the LFOT, which partitions the mechanical parameters into those governing the airway (Raw and Iaw) and tissue compartments (G, H, and {eta}), we have shown that the improvements in lung compliance observed previously (4) are accompanied and preceded by a marked decrease in tissue resistance and a consequent change in the mechanical coupling of the viscous and elastic forces within the lung tissue. The changes in lung function were strongly associated with the increase in the alveolar surfactant pool size.

Morphometric Findings

The parenchymal fraction (alveolar air space, alveolar ducts, and alveolar walls) was the main lung tissue component (78%) in control animals and accounted for an even greater fraction of the lung tissue in lambs exposed antenatally to IA endotoxin for a period of at least 4 days (85–88%) due to a reduced contribution from the nonparenchyma (conducting airways, perivascular tissue, intersitium, interlobular septa, and the pleura). Within the parenchymal compartment, there was an early (2-day group) increase in the contribution of the alveolar duct air space to the parenchymal compartment at the expense of the alveolar air space. Thinning of the parenchymal tissue was a late phenomenon coinciding with marked improvements in both oxygenation and ventilation (4). Although we did not observe a significant reduction in total alveolar number within the limited number of animals reported in Table 1, when this analysis was extended to include morphometry from those animals in which oscillatory impedance was not measured, a strong trend toward decrease in total alveolar number (P = 0.078) and alveolar volume (P = 0.087) was noted from as early as day 2 after IA endotoxin injection (data not shown). These trends are consistent with the previous observation by Moss et al. (7) of the effect of early gestational exposure to endotoxin and that of Willet and colleagues (13) in animals exposed 7 days before delivery.

Forced Oscillatory Mechanics

With the use of one-way ANOVA, significant changes in dynamic compliance (calculated from the ratio of VT/kg and the difference between peak inspiratory and positive end-expiratory pressures) in animals receiving IA endotoxin injection at least 7 days before delivery at 125 days were observed previously (4); this is represented in Fig. 3 to provide a benchmark for the significance of the results of the forced oscillatory mechanics. When ANOVA of the dynamic compliance was limited to the subset of animals able to be studied with LFOT, significance of the change in dynamic compliance was only observed in the group given an IA endotoxin injection 15 days before delivery in agreement with our measurement of tissue elastance (1/compliance). In contrast, we observed a strong trend toward a decrease in tissue viscoelasticity as early as 2 days and tissue damping within 4 days after IA endotoxin injection, illustrating the potential of this technique to provide sensitive measurements of lung function.

The LFOT was used previously in the preterm lamb to demonstrate changes in partitioned lung mechanics with advancing gestation (8), following antenatal betamethasone (8) or prophylactic surfactant administration (9), and during a volume recruitment procedure in lambs ventilated with high-frequency oscillatory ventilation (9). The dominant impact of tissue impedance on overall respiratory impedance has been a consistent feature in the control lambs for each of these investigations. A reduction in tissue impedance was the most notable effect of advancing gestation, antenatal steroids, prophylactic surfactant administration, and volume recruitment. The current investigations further support the dominance of tissue impedance in the preterm lung.

Although both tissue damping and elastance decreased after IA endotoxin injection with increasing exposure duration, there was a more marked reduction in tissue damping, leading to a decrease in tissue {eta}. Altered {eta} implies a change in the balance of viscous (dissipative) and elastic properties of the lung. In healthy lungs, tissue hysteresivity is a species-independent constant (2), although it may be increased in the immature preterm lamb (8) and before volume optimization with either recruitment maneuvers or exogenous prophylactic surfactant administration (9).

In animals exposed to IA endotoxin injection, mean airway pressure decreased with increasing exposure time, consistent with the previously reported timing of increase in surfactant pools and ventilation efficiency index (4). To facilitate measurements of impedance at physiologically appropriate lung inflation pressures, we performed the forced oscillatory measurements at the prevailing mean airway pressure as determined from the ventilator settings 35 min after delivery. At these pressures, the airways are likely to be splinted open (6), and hence it is not surprising that we did not observe significant differences in Raw between treatment groups.

Comparison of Lung Function, Clinical, and Morphometric Indexes

The sensitive technique for measuring tissue mechanics used in this study identified that changes in lung function occur sooner after IA endotoxin injection than reported previously (4). Although there were significant associations between each of the indexes of tissue impedance with a number of morphometric indexes on bivariate analysis, the most significant factor contributing to the variability in tissue impedance on stepwise linear regression was the log alveolar saturated phosphatidylcholine (Sat PC) pool size, and to a lesser extent the tissue fraction. Both of these factors are likely to act by increasing lung volume. If estimated lung volume at the prevailing distending pressure during measurements was included in the regression model, then 93.3% of variability in tissue damping and 91.2% of the variability in tissue elastance could be explained. The increased accountability of H by including estimated lung volume at measurement may reflect additional contribution from nonsurfactant factors such as morphometric indexes influenced by a uniform fixation pressure.

Because tissue damping was influenced by alveolar Sat PC pool size to a greater degree than tissue elastance, the alveolar Sat PC pool size was the only significant factor contributing to tissue {eta}. Changes in surface tension have been proposed as one mechanism whereby the {eta} of the lung tissue may be altered (2), which was confirmed recently in preterm lambs receiving prophylactic surfactant at delivery (9). Although no other significant factors contributing to variability were observed, it is worth commenting on the extremely low {eta} values for the animals exposed to IA endotoxin for the 15-day group compared with previously published species-independent values for the healthy lung (2). Although low {eta} values may indicate relative overinflation of the lung occurring despite lower mean distending pressures during the measurements (11), lung volumes estimated from the pressure volume curve at which forced oscillatory measurements were obtained did not suggest overinflation. The low {eta} values could also be indicative of a dysmaturation process in which lung remodeling following an inflammatory insult plays an important role. Integrity of the interrelationships between intraparenchymal connective tissue components, surface film, and contractile elements of the lung tissue may be of critical importance in achieving necessary synchronous expansion of the alveolar ducts in the healthy lung (2). Dysmaturation could contribute to a mechanical uncoupling of the dissipative and elastic forces within the lung tissue, indicating loss of this integrity. The marked decrease in parenchymal tissue fraction and relative prominence of alveolar ducts in those animals having the longest exposure to endotoxin support this possibility.

Conclusions and Clinical Relevance

We have shown that significant changes in lung mechanics at delivery can be observed within 4 days of IA endotoxin injection, reflected initially as a decrease in tissue damping, and accompanied later by concomitant reduction in tissue elastance. Despite the inflammatory and structural changes that follow such exposure, the increase in alveolar Sat PC pool size and, to a lesser extent, the decrease in parenchymal tissue fraction were identified as the most important associations with reduced oscillatory tissue impedance after IA endotoxin. These findings are consistent with the absence of significant respiratory disease at birth in infants where there was histological evidence of chorioamnionitis (12) and will be important to the interpretation of changes in forced oscillatory mechanics in the newborn infant.


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The authors gratefully acknowledge financial support from National Institute of Child Health and Human Development Grant HD-12714, the Hungarian Scientific Research Fund Grant T42971 [GenBank] , and the Women's and Infants' Research Foundation. Dr Pillow was supported by a National Health and Medical Research Council (Australia) medical postgraduate scholarship during the period that the studies were undertaken.


    ACKNOWLEDGMENTS
 
The authors acknowledge the assistance of Dr. James Padbury, Dr. Gore Ervin, and the staff of the Medina Agricultural Station for assistance with the care of the animals used in this study and participation in the day-to-day study activity.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. J. Pillow, c/o Neonatal Clinical Care Unit, King Edward Memorial Hospital, Bagot Road, Subiaco 6008, Perth, Australia (E-mail: janep{at}ichr.uwa.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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