Mechanical dissociation of bronchi from parenchyma in the immature piglet lung

Departments of Pediatrics and Medicine, Brown University, ¹ Rhode Island Hospital and ² Memorial Hospital, Providence, Rhode Island 02902

	ABSTRACT

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Previous studies of isolated piglet lungs suggested that local distending forces around bronchi might be relatively weak before postnatal growth and maturation. The present study used tantalum bronchograms to compare pressure-diameter relationships of bronchi in situ and after excision from the parenchyma in immature (3- to 7-day-old) and mature (3-mo-old) piglets. The mature group reproduced behavior that is well established in mature lungs from other species; i.e., bronchial diameters maintained a constant relationship to the parenchyma as the lungs were deflated from maximum to minimum volume. In sharp contrast, diameters failed to change until the immature lungs were deflated to <5 cmH₂O transpulmonary pressure. Total percent change in bronchial diameter was then only 24% in the immature lungs compared with 47% in the mature lungs (P < 0.002). Total elastances of mature generation 3-8 bronchi did not change when they were excised from the parenchyma. However, in the same generations of immature bronchi, total elastances were lower after than before (1.06 vs. 1.60 cmH₂O/%, P < 0.05) excision from the parenchyma. Elastances of the excised immature and mature bronchi were then the same (1.06 vs. 1.03 cmH₂O/%, not significant). Because elastic moduli of the lung parenchyma are also similar in the two age groups, it was concluded that local features of airway-parenchyma coupling limited the generation of local parenchymal recoil around bronchi in the immature lungs.

lung growth; airway-parenchyma interdependence; bronchograms

	INTRODUCTION

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A PREVIOUS STUDY DOCUMENTED remarkable behavior of extra-alveolar blood vessels during inflation of excised immature piglet lungs (23). Lung inflation compressed these vessels, in contrast to the decompression that had been observed consistently during inflation of excised mature lungs from several species (1, 13, 17, 29, 33). The new observation suggested transmission of alveolar pressure (PA) to the vessels as the lungs were inflated, possibly reflecting relatively weak distending forces in the tissue connections to the vessels.

The present study examines the effects of lung inflation on bronchi in these immature, excised lungs. Previous work has suggested that diameters of bronchi track movement of the surrounding parenchyma closely as excised, mature dog (6, 20, 32) and human (28, 37) lungs are inflated and deflated. Hughes et al. (10) suggested that adherence of various-sized airways to this behavior requires a sufficient mechanical association with the parenchyma to generate local distending forces that equal or exceed lung recoil pressure. On the basis of our findings with blood vessels, we hypothesized that local distending forces around bronchi might be relatively weak in immature lungs near full inflation. As a consequence, bronchial diameters might fail to fully expand.

The work reported here further characterizes postnatal lung development in the piglet, which is a model that has been studied by morphometry (7, 21, 30, 38) and mechanics (21, 22). Present results show that internal diameters of bronchi in the immature piglet lung can dissociate from parenchymal expansion within the operative range of lung volumes in vivo. This finding pertains to current concepts linking bronchial reactivity to the cycling forces of breathing, particularly as applied to long-standing evidence for bronchial hyperreactivity in immature animals and human infants.

	METHODS

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Animal model. For purposes of the present study, piglet lungs were large enough at birth to allow mechanical and radiological studies. Two groups of female Duroc piglets were studied: one 3-7 days of age (1.3-2.1 kg body wt), which was designated the "immature" group, and the other 3 mo of age (25-40 kg body wt), which was designated the "mature" group. Age of the immature group was chosen to avoid temporary characteristics of structure and function in freshly newborn piglet lungs (23). Age of the mature group was chosen to avoid the practical difficulties in handling large, fully grown pigs. Although the piglet reaches sexual maturity at ~9 mo of age (25), previous morphological studies (7) have shown that changes in pulmonary vessels and parenchyma during the first 3 mo are similar to those that occur in the human through adolescence.

All studies were approved by the Animal Welfare Committee of Rhode Island Hospital.

Surgical and mechanical techniques. Each piglet was anesthetized with ketamine (100 mg/kg im) with 10% acepromazine and was also given atropine (0.04 mg/kg im) before being killed by an intravenous overdose of pentobarbital sodium. An endotracheal tube was introduced by tracheostomy and secured. The chest cavity was approached from the abdomen by a midline ventral incision and opened.

Static expiratory pressure-volume (P-V) curves were obtained in the open-chest piglet by stepwise deflation of the lungs with a volume-displacement apparatus while airway pressure was monitored. In the mature group, P-V curves were obtained from the left lower lobe only. Transpulmonary pressure (PL) was taken as airway pressure minus pleural surface pressure, which was ambient. Lung or lobe volume at 0 cmH₂O PL was measured by water displacement, and gas volume was calculated by subtracting lung or lobe weight in grams, where tissue specific gravity was assumed to be 1. Absolute lung or lobe volume was then calculated by adding gas volume at 0 cmH₂O PL to the changes measured by the volume-displacement apparatus. The final P-V result was taken as the mean of three curves.

After removal from the thorax, the lungs were dissected free of the heart and used for studies of bronchial behavior in situ (see below) or for studies of excised bronchial behavior, as follows.

Major bronchi were excised from the parenchyma in three pairs of lungs from each group. The bronchi were approached by separating intralobar subsegments with a cotton pledget. Side branches, which constitute lobar or segmental bronchi, were occluded by plastic plugs, cyanoacrylate glue, and sutures, with care taken to leave the main trunk of the evergreen-like bronchial tree (Fig. 1). This trunk was either lined with tantalum and studied radiologically or used for P-V relationships as follows.

View larger version (114K): [in this window] [in a new window]   Fig. 1.   A: tantalum bronchogram of 5-day-old piglet lung at 5 cmH2O transpulmonary pressure (PL). B: parenchyma has been excised and trunk inflated to 5 cmH2O transmural pressure (Ptm).

P-V relationships (n = 3 in each group) were determined in a preparation that was obtained by tying a catheter into the bronchial trunk. The preparation was verified to be airtight by inflation under normal saline and was then suspended vertically in air and connected to a calibrated syringe. Changes in volume were monitored by a linear displacement transducer mounted to a Harvard pump, which drove the barrel of the syringe. The airway path was cycled between 3 and 30 cmH₂O over 4-6 min per cycle until three reproducible quasi-static P-V relationships had been recorded. Absolute volume of the airway path at 0 cmH₂O transmural pressure (Ptm) was then obtained by underwater displacement with use of methods identical to those described above for absolute lung volume.

Bronchial Ptm-diameter relationships were estimated by correcting the P-V curves of the excised bronchi for gas compression and changes in length. Gas compression was measured by cycling the apparatus dead space plus bronchial trunk volume (represented by an occluded syringe) between 3 and 30 cmH₂O. Changes in length were estimated from radiological studies at various levels of Ptm. Actual lengths of the bronchial trunks were measured at 0 cmH₂O Ptm and used to calculate diameters of assumed uniform cylinders.

Radiology. A catheter was wedged distally near the tip of the left lower lobe and then tied into place around the left main stem bronchus. The lung was then suspended in a vacuum jar and sealed with the catheter open to the outside. Tantalum powder (325 mesh) was insufflated into the catheter as the lung was inflating during application of 15 cmH₂O to its pleural surface. This procedure was done two or three times in the immature lungs and three or four times in the mature lungs, with the catheter moved proximally before each insufflation to image side branches of the left lower lobe bronchus.

After insufflation of tantalum, the lung was removed from the vacuum jar, and a catheter was tied into the main lobar bronchus and connected to a water trap for delivery of constant airway pressures, as monitored by a pressure transducer. Radiographs of the lung were taken at airway pressures of 30, 25, 20, 15, 10, 5, 2, and 0 cmH₂O. The lung was reinflated to 25 cmH₂O briefly before each deflation to the new PL. After the radiographs at 30, 25 and 20 cmH₂O, the lung was taken down and degassed to prevent gas trapping over the 15- to 20-min procedure.

Source-to-film distance was 105 cm for the immature lungs (object-to-film distance 2-3 cm) and 210 cm for the mature lungs (object-to-film distance 4-5 cm). A high-resolution film (Dupont Chronex Microvision) and image intensifying screen (Quanta V) were used. Energy settings were typically 60 kvp and 6 mA for the immature lungs and 60 kvp and 17 mA for the mature lungs.

Photographic enlargements were made from the radiographs. Diameters of bronchi were measured using a Bausch and Lomb measuring magnifier with divisions of 0.1 mm. Four or five sites for measuring were chosen on the basis of relative location (generation number) and consistency of diameter. Bronchial generations were designated according to the Weibel terminology (36), where the trachea is generation 0 and each peripheral branch along any path is one generation higher (Fig. 1). Sites measured in this study were located in generations 3-11. The reader was blinded to the PL corresponding to the various radiographs.

Excised airway trunks (n = 3 in each group) were coated internally with tantalum and tested for pressure-diameter relationships in six lungs from the immature group and seven from the mature group. A radiograph was taken after each of eight levels of Ptm (between 30 and 0 cmH₂O) had been maintained for 30 s. The object-to-film and source-to-film distances were the same as those used as for the studies in situ. Additional radiographs were taken at 0, 2, 5, 10, and 25 cmH₂O Ptm on the inflation limb of the pressure-diameter cycle in two lungs from each group. The technique for measurement of diameters was identical to that for the studies in situ, although only sites in generations 3-8 could be used (Fig. 1).

Statistical methods. Results from the two groups were compared using the two-tailed, unpaired t-test, where P < 0.05 was assumed to indicate significance.

	RESULTS

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Following convention, pressure-diameter behavior of bronchi is expressed as elastance: change in pressure  change in diameter. Elastance of intact (in situ) bronchi is termed effective elastance. Because diameters of bronchi varied widely within each group and between the two experimental groups, change in diameter is always characterized as percent change. The standard denominator for these comparisons was arbitrarily chosen as diameter at 5 cmH₂O Ptm or PL.

Bronchial diameters in situ. At 5 cmH₂O PL, internal diameter of the left main stem bronchus (generation 2, Fig. 1) was ~13 mm in the mature group and 6 mm in the immature group. Diameters along the main bronchial trunk were substantially larger than those of the side branches in both groups. In the mature lungs, the large-diameter segments along the main trunks had slightly higher effective elastances than the smaller bronchi that constituted the side branches (P < 0.05). Such a relationship between absolute diameter and effective elastance could not be demonstrated in the immature lungs.

The immature and mature lungs were compared first for side branches from the main trunks, including any daughter branches that were comfortably within resolution of the radiographs (Fig. 1). At low PL during lung deflation, the immature bronchi underwent slightly larger percent changes in diameter than did the mature bronchi (Fig. 2). Between 2 and 0 cmH₂O PL, for example, diameters of generation 4 bronchi decreased by 13% in the immature lungs vs. 7% in the mature lungs (P < 0.05). However, the outstanding result was at higher PL, where the immature bronchi underwent much smaller percent changes in diameter. Between 30 and 5 cmH₂O PL, the diameters of the immature bronchi did not change significantly, whereas the diameters of the mature bronchi decreased by 25%. Thus effective elastances in the immature lungs were slightly lower at low PL but much higher at >5 cmH₂O PL than in the mature lungs. Total percent change in diameter between 30 and 0 cmH₂O PL was much smaller in the immature lungs (24%) than in the mature lungs (47%, P < 0.002). Furthermore, diameters in the immature lungs were at 95% of their maxima at 3.6 ± 1.2 (SD) cmH₂O PL (vs. 10.8 ± 3.2 cmH₂O in the mature lungs, P < 0.001).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Deflation pressure-diameter (D) curves in situ for generation 3-11 bronchi in immature and mature lobes. Diameters are represented as ratios to measurements made at 5 cmH2O PL. Error bars, SE.

Changes in bronchial diameter were compared with changes in the cube root of absolute lobe volume (V^1/3) during deflation in the two groups (Fig. 3). In the mature group, bronchial diameter tracked V^1/3 closely throughout lung deflation. In the immature group, V^1/3 decreased much more than bronchial diameter until the lungs were at 2 cmH₂O PL, near minimum volume.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Changes in diameters of generation 3-11 bronchi compared with the cube root of air space volume (V1/3) during lung deflation. Diameters and V1/3 are represented as ratios to measurements made at 5 cmH2O PL. , Immature lobes (n = 5); , mature lobes (n = 6); dashed line, identical ratios; error bars, SE.

Excised bronchi. Necessities of dissection limited measurements in both groups almost exclusively to the relatively large-diameter main trunks (generations 3-8, Fig. 1). With the trunks at maximum inflation, ideal diameters calculated from P-V studies correlated well with the average of radiographic diameters along the excised trunks in both groups. At lower levels of Ptm, however, pressure-diameter behavior of the excised trunks depended heavily on their inflation/deflation histories. More inflation/deflation hysteresis occurred during the static radiographic studies than during the quasi-static P-V studies. As a result, diameters low on the deflation limbs of the static radiographic curves were larger than those calculated at similar levels of Ptm from the quasi-static P-V curves. This observation is consistent with those from two previous radiographic comparisons of intact and excised bronchi (11, 26), in which diameters were larger in the excised than in the intact preparations.

Percent change in diameter vs. Ptm during deflation was the same in the excised immature and mature bronchi, whether results were derived from radiographic or P-V studies. At both ages, bronchial elastance was much higher during the static radiographic studies than during the quasi-static P-V studies (Table 1) because of the hysteresis mentioned above. Diameters in millimeters calculated from the quasi-static P-V loops are shown in Fig. 4.

View this table: [in this window] [in a new window]   Table 1.   Total elastances of generation 3-8 bronchi during deflation from 30 to 0 cmH2O airway pressure in situ and after dissection

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Pressure-diameter curves in situ and after excision from parenchyma for generation 3-8 bronchi from mature (top, n = 3) and immature (bottom, n = 3) lobes. Deflation curves in situ (solid lines) are compared with inflation/deflation loops (dashed lines) after excision. Error bars, SD.

Comparison of bronchi in situ vs. excised. Direct, site-for-site comparisons from radiographs were too sparse for any conclusions about absolute diameters in situ vs. excised. In addition, it was assumed that the high elastances of the excised bronchi were artifacts caused by the static radiographic techniques. Diameters calculated from the deflation limbs of the quasi-static P-V loops were compared with diameters measured in situ during lobe deflation (Fig. 4, Table 1).

In the mature group, total percent change in diameter between intrabronchial pressures (Pbr) of 30 and 0 cmH₂O did not differ in the two conditions, but changes were smaller in situ than after excision in the immature group (P < 0.05). Matching of diameters from the two conditions in Fig. 4 assumes that bronchi-parenchyma interdependence was negligible (i.e., Pbr = Ptm) at 5 cmH₂O Pbr. This assumption draws some support from a previous study of excised dog lungs (31), in which pressure around relaxed bronchi (peribronchial pressure, Px) was calculated to equal pleural pressure at lung volumes <60% of maximum.

Estimation of local parenchymal recoil. Local parenchymal recoil (LPR) is defined as the pressure difference across parenchymal attachments to the bronchus (LPR = PA  Px) and is balanced by bronchial wall recoil (BWR = Pbr  Px; Fig. 5). Because Px is inaccessible, neither LPR nor BWR can be measured directly in situ. An estimate of LPR (32) is available from direct measurement of excised BWR, in which Px = 0, and the assumption that LPR = BWR at the same bronchial diameter in situ. This methodological assumption does not involve any prescribed notion of bronchus-parenchyma coupling. In contrast, another estimate (14, 18) assumes that LPR is essentially overall lung recoil (PL) modified by distortion at the bronchus-parenchyma boundary (see APPENDIX).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Schematic drawing of a bronchus end-on surrounded by alveolar walls. Arrows, tissue (solid) and gas (open) forces that widen and narrow the bronchus; Pbr, intrabronchial gas pressure; Px, peribronchial interstitial pressure; PA, alveolar gas pressure; BWR, bronchial wall recoil; LPR, local parenchymal recoil.

The two estimates of LPR, as plotted against lung volume in Fig. 6, yield similar results in the mature group but differ sharply in the immature group, in which the estimate from BWR reaches a limit between 6 and 7 cmH₂O.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   LPR around bronchi as a function of lung volume during deflation of mature (A) and immature (B) lungs. (D'  S'), measure of distortion; µ, shear modulus; dashed lines, predictions from excised pressure-diameter curves (Fig. 4) combined with elastic moduli from the parenchyma (see APPENDIX); solid lines, values calculated from comparison of pressure-diameter curves in situ and after excision (Fig. 4).

	DISCUSSION

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Comparisons with previous studies of other species. Pressure-diameter curves of bronchi in situ have been measured previously in lungs excised from mature dogs (11, 24, 26, 31, 32) and humans (28, 37). In agreement with those studies, bronchial diameters from our 3-mo-old piglet lungs showed convincing plateaus near maximum lung inflation and were 30-40% smaller at 0 cmH₂O PL. Effective elastances of the larger bronchi were higher than those of the smaller bronchi, as reported previously in dog (26) and human (28) lungs.

Bronchial diameters tracked the cube root of air space volume as our 3-mo-old piglet lungs were deflated. In previous studies of excised human (28, 37), dog (6, 10), and cat (12) lungs, this pattern of geometric similarity was interpreted as evidence for close matching of bronchial and parenchymal elastances. However, Brown and Mitzner (2) noted the absence of geometric similarity between the behavior of airways and air spaces in serial computerized tomography scans of pharmacologically relaxed dog bronchi in vivo. They observed a critical PL (~5-7 cmH₂O) above which bronchial elastance greatly exceeded parenchymal elastance and below which they found the opposite relationship. As an explanation, they cited the putative behavior of epithelial basement membranes, which fold and unfold easily at low Ptm but become virtually indistensible when completely unfolded at higher Ptm. In this case, our excised bronchi should have shown sharply biphasic pressure-diameter relationships; however, transitions to maximum diameter were far from abrupt (Fig. 4). The difference in results might be caused by the extensive handling and instrumentation of the bronchi during our study, or it might reflect a real mechanical difference between dog and pig bronchi.

New findings in the immature group and possible sources of error. The immature bronchi in situ stood out among the various comparisons; they were relatively stiff in diameter changes compared with mature bronchi, in situ or dissected, or compared with themselves after dissection (Table 1). As a control, dissection of the mature bronchi from the parenchyma had no effect on their total elastances. The distinctive behavior of the immature bronchi in situ was characterized by failure to narrow until low PL results were reached during lung deflation.

Excised lungs are prone to interstitial emphysema that may not be obvious from their gross mechanical behavior. Interstitial air could dissociate bronchi from the surrounding parenchyma, explaining the unusual pressure-diameter behavior as an artifact. Because immature rat lungs rupture at lower distending pressures than mature rat lungs (27), it is reasonable to suspect that our immature piglet lungs were especially prone to disruption. A few of our excised mature and immature lungs developed obvious air leaks, as shown by their failure to hold constant airway pressures, and were excluded from the study. The possibility of more occult interstitial emphysema was addressed in passing during stepwise inflation to 30 cmH₂O PL as serial high-resolution computerized tomograms were made (unpublished data). Because these tomograms showed no evidence of air leaks, we are confident that our findings are not explained by peribronchial interstitial emphysema.

Although the excised immature and mature bronchi were indistinguishable in terms of percent change in diameter during deflation, it is conceivable that different degrees of bronchomotor tone existed in situ, despite the pretreatment of all animals with atropine. Increase in tone reduces diameters of dog bronchi in vivo much more at low than at high PL, so that percent change in diameter during deflation increases as the bronchoconstricted lung is deflated (2). Percent changes in diameter were very similar in our two groups at <5 cmH₂O PL in situ (Fig. 3). Therefore, large differences in bronchomotor tone are unlikely, and we find no reason to believe that the similarity of pressure-diameter characteristics found in the excised bronchi did not exist in situ.

Significance of results in immature lungs. On the basis of our previous studies of extra-alveolar arteries in the same two age groups of piglets (23), we hypothesized that local distending forces around bronchi might be relatively weak near full inflation in the immature group. As estimated from pressure-diameter relationships of the bronchi in situ and after dissection, LPR reaches a limit of 6-7 cmH₂O in the immature lungs (Fig. 6), supporting the hypothesis.

Can the limits of LPR in the immature lungs be explained by limits of overall parenchymal recoil? During postnatal growth and maturation, a shift in the overall P-V relationship of human or pig lungs occurs, which, according to the method of comparison, is described as an increase in elastic recoil (3), a decrease in bulk modulus (22), or an increase in half-inflation pressure (20). Such a shift is apparent in Fig. 3, in which the immature lungs show relatively small percent changes in volume between 30 and 5 cmH₂O PL. This feature of parenchymal maturation was assessed as an explanation for our findings by assuming that LPR is essentially overall lung recoil as modified by distortion at the bronchus-parenchyma boundary (see APPENDIX). These assumed values matched measured LPR reasonably well in the mature group but not in the immature group, in which they grossly overestimated LPR (Fig. 6). Thus it appears that limits on LPR in the immature lungs are imposed at the bronchus-parenchyma boundary. Limitation of outward distending forces transmitted from the parenchyma could be imposed by local features of airway-parenchyma coupling, such as increased wall thickness (14, 19), fluid shifts centrally along the peribronchovascular sheath, or reduced density of parenchymal attachments to the sheath.

LPR constitutes an elastic load that opposes bronchoconstriction (4, 9, 19) and is the motive force of bronchodilation. Mature dog lungs can generate LPR pressures near 10 cmH₂O around maximally constricted bronchi, even at volumes below functional residual capacity (31). Therefore, limitation of LPR to 6-7 cmH₂O in immature lungs could amplify bronchoconstriction and undermine its reversal by deep breaths. In addition, force fluctuations that determine the dynamic equilibrium of actin-myosin binding are linked closely to LPR (5). Human infants are known to have relatively strong physiological responses to bronchoconstrictor agents and weak responses to bronchodilator agents (8, 16, 34, 35). The present study offers one explanation for these observations in terms of LPR and justifies a morphological approach to the maturation of airway-parenchyma coupling.

	APPENDIX

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

A continuum mechanics analysis previously applied to vessel-parenchyma interdependence in piglet lungs (23) is modified to predict LPR around the bronchi. The analysis is applied to known pressure-diameter characteristics of the excised bronchi and to previously measured values for elastic moduli of the surrounding parenchyma (20). The bronchus is assumed to be circular in cross section, and wall thickness is considered negligible. If D₀ represents the diameter of the bronchus at 0 Ptm, then any other diameter can be expressed as a strain by the ratio D' = D/D₀. Local strain in the tissue attachments around the bronchus is assumed to be the same as average parenchymal strain at the measured lobe volume. Average parenchymal strain (S) is defined as a linear dimension of absolute lung volume (V) relative to a linear dimension of a reference volume (V₀); that is, S' = (V/V₀)^1/3, where V₀ is the lobe volume at 0 PL. Thus the strains D' and S' have been scaled to be equal under reference conditions in which Ptm = 0 and PL = 0.

If D' = S', the tissue attachments are expanded to the same strain as the parenchyma elsewhere and LPR = PL. When the parenchyma is distorted around the bronchus, D' does not equal S' and D'  S' can be used as a measure of the distortion. When D' < S', the attachments are strained beyond S'; therefore, LPR > PL. According to the linear elasticity analysis suggested by Lambert and Wilson (15), the increase in LPR depends on the parenchymal shear modulus (µ) and the fractional distortion of the parenchymal attachments. Because D' and S' are fractional deformations, D'  S' can be applied to the linear elasticity analysis, which becomes
When D' > S', the attachments are relaxed to a strain smaller than S'; therefore, LPR < PL. However, we consider the parenchymal attachments to be unable to carry a compressive force; that is, LPR must be >0. The above linear elasticity equation predicts LPR = 0 when D' > S' + PL/2µ.

	FOOTNOTES

Address for reprint requests and other correspondence: A. L. Mansell, Rhode Island Hospital, SWP 4, 593 Eddy St., Providence, RI 02903 (E-mail: anthony_mansell{at}brown.edu).

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. §1734 solely to indicate this fact.

Received 23 September 1999; accepted in final form 10 February 2000.

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TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

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