INTRODUCTION

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ALVEOLAR MICROVESSELS are subjected to a range of perfusion and inflation pressures that affect their functional diameters. In a recent report (2), our laboratory showed in zone I, where inflation pressure exceeds perfusion pressure, that these microvessels were compressed but not obstructed. In a subsequent report (3), we estimated the functional diameter of these vessels in zone I to be 1.7 µm. When we raised the pulmonary arterial pressure (Ppa) to equal the inflation pressure (zone I/II border), we estimated that the microvessel diameter would increase to 6-8 µm (4).

In those previous studies, all lungs were inflated to near-total lung capacity (25 cmH₂O). Ppa was set to either 20 cmH₂O (zone I) or 25 cmH₂O (zone I/II border) (14). Results of those studies showed that a rise in Ppa from 5 cmH₂O below inflation pressure to a value equal to inflation pressure caused the functional diameter to rise by 4-6 µm.

We also found in those studies that the fraction of alveoli containing particles rose with the Ppa. We found that 13% of alveoli contained 1-µm-diameter particles in zone I, but this increased to 27% at the zone I/II border. Thus both the fraction of perfused alveoli and the microvessel diameter rose with Ppa.

In this report, we describe results of perfusing lungs in zone II. As in our previous studies (2-4), the lungs were inflated to 25 cmH₂O. However, in these studies, Ppa was set to 30 cmH₂O so that it exceeded inflation pressure by 5 cmH₂O. In addition, we also measured for the first time the particle concentrations in the venous effluent. We used these data as an index of the size of the particles trapped within specific microvascular segments.

	METHODS

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Animal preparation. Eighteen male rats (450-550 g) were anesthetized with intraperitoneal ketamine (40 mg/kg), xylazine (6 mg/kg), and acepromazine (1 mg/kg) and placed in a supine position. A polyethylene cannula (PE-60) was inserted into the femoral vein, and heparin (750 U/kg) was injected through it. After 10 min to allow the heparin to circulate, we severed the femoral artery and let the animal exsanguinate. The shed blood volume was recorded minute by minute during exsanguination, and an equal volume of Ringer lactate was infused to facilitate the washout of red blood cells from the circulation. This improved subsequent perfusion of the isolated lungs. The volume of Ringer lactate infused equaled the volume of blood shed and averaged 25-35 ml (5-6% of the animal's body wt). We collected blood and infused Ringer lactate until the animal's spontaneous breathing ceased.

Histology. The lungs were removed from the isopentane and placed into liquid nitrogen where they were cut, by using a scalpel, into blocks of ~7×7×7 mm. Ten blocks, representing ~30% of the total lung volume, were randomly selected and placed into dehydrated, chilled (70°C) ethanol. The blocks were stored in a freezer (70°C) for 3-5 days to dehydrate the tissue by freeze substitution. After being warmed (4°C), the ethanol was replaced with unpolymerized embedding resin (JB-4; Polysciences), and the blocks were placed under vacuum to remove residual air. The blocks were then embedded in polymerized resin. We cut three to four sections (75-µm-thick) from each block using a heavy-duty microtome (Reichert Ultracut) and mounted the sections unstained on glass slides.

View larger version (78K): [in this window] [in a new window]   Fig. 1.   Confocal optical sections from lungs perfused with 4- (A) and 5-µm-diameter (B) fluorescent latex particles. The 4-µm-diameter particles (arrows, A) were able to enter septal vessels under zone II conditions, but 5-µm-diameter particles (arrow, B) were not. These results are consistent with our hypothesis that septal microvessel diameter in these conditions lies between 4 and 5 µm. Optical section thickness (A and B) is 0.3 µm; scale is 25 µm. A lies within alveolar septal plane; B lies within air space above septal plane. See Fig. 2 for three-dimensional reconstructions of A.

Venous particle concentrations. Latex particle concentrations in venous effluent were measured by using a flow cytometer (FACScan, Becton Dickinson). The cytometer was calibrated so that each latex perfusate produced 2,000-4,000 forward and side-scatter events per sample, corresponding to one event per particle. Particle events in venous samples were then measured and expressed as fractions of those in the perfusate. Five venous samples were measured from each perfused lung. Results were averaged among all lungs perfused with particles of each diameter.

Statistics. Data obtained for particles of each diameter were pooled and expressed as means ± SD. The number of measurements made and the number of alveoli studied are shown in Tables 1 and 2, respectively. Results among particle diameters were compared by using one-way analysis of variance. Post hoc comparisons were performed by using Fisher's paired least-significant difference test (version 1.03, StatView; Abacus Concepts, Berkeley, CA). Differences were considered to be significant at P  0.05. 

	RESULTS

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Perfusate flows. During 10 min of perfusion with Ringer lactate before latex perfusion, flows averaged 7.2 ± 3.0 ml/min. During the 10 min of subsequent latex perfusion before freezing, flows averaged 6.3 ± 3.4 ml/min. Flows did not differ significantly among lungs perfused with particles of each diameter.

Particle densities. Average particle densities measured within septa ranged from 0.068 ± 0.024 to 0.015 ± 0.007 particles/µm² for 1.0- and 4.0-µm-diameter particles, respectively (Table 2). We found no examples of 5-µm-diameter particles within septa, although we did see examples of these particles within corner vessels (Fig. 1). Densities for particles of any one diameter differed significantly from densities of the others (P  0.05).

Fraction of septa with latex. In lungs perfused with 1.0-µm-diameter particles, the fraction of septa found to contain latex was 0.45 ± 0.12 (Table 2). This fraction fell to 0.31 ± 0.12 and 0.25 ± 0.09 for the 2.0- and 4.0-µm-diameter particles, respectively. The 5.0-µm-diameter particles were absent from septa.

Venous particle concentrations. Particle concentrations in venous effluent, expressed as fractions of the concentrations in the arterial inflow, were 0.54 ± 0.28 and 0.67 ± 0.32 (not significant) for lungs perfused with 1.0- and 2.0-µm-diameter particles, respectively (Table 1). The venous concentration ratios dropped to 0.022 ± 0.026 and 0.004 ± 0.003 for lungs perfused with 4.0- and 5.0-µm-diameter particles, respectively. Particles of 6 and 10 µm diameter were absent from the venous effluent.

	DISCUSSION

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Our data show that, in the zone II conditions under which the lungs were perfused, particles with diameters of 1.0, 2.0, and 4.0 µm entered septa, whereas 5.0-µm-diameter particles did not. However, 5.0-µm-diameter particles entered corner vessels adjacent to septa, whereas 6.0-µm-diameter particles did not. These results suggest that, under these conditions, the largest alveolar septal microvessels had a functional diameter >4.0 but <5.0 µm and further suggest that corner vessels had diameters between 5.0 and 6.0 µm.

This conclusion is supported by the venous effluent concentrations, which were near zero for the 5.0-µm-diameter particles and at zero for the 6.0-µm-diameter particles. Concentrations were only 0.022 ± 0.026 for the 4.0-µm-diameter particles, suggesting that 4.0-µm-diameter particles were trapped within septal vessels, whereas 5.0-µm-diameter particles were trapped within corner vessels. The 1.0- and 2.0-µm-diameter particles were partially trapped by septal vessels, based on their venous concentrations, but were able to flow through the lungs with much less restriction than the larger particles could.

Our results also suggest that no significant shunt pathway around the corner vessel-septal vessel network was present within these lungs. If a shunt pathway existed, we would have expected higher venous concentrations of particles with diameters of 5.0 µm and larger.

Our results can be better appreciated by comparing them with the results of our previous studies conducted in zone I and at the zone I/II border (3, 4). In those studies, the lungs were inflated to the same pressures as those described here (25 cmH₂O) but were perfused at Ppa of 20 or 25 cmH₂O, respectively. The latex concentrations in the perfusates and the number of minutes the lungs were perfused are identical to the values in our present study. Therefore, it is reasonable to compare results of those studies with our present one. Particle densities and the fraction of septa that contained latex in the previous studies are shown in Table 3.

Comparing septal densities for particles of common diameter among the studies shows that densities were higher in zone II than either at the zone I/II border or in zone I. Because all lungs were inflated to the same pressure, these results suggest that the septal capillary diameter increased with the vascular pressure. For example, particles with diameters of 1.0 µm had septal densities of 0.068 ± 0.024 particles/µm² in zone II (Table 2), 0.038 ± 0.013 particles/µm² at the zone I/II border (Table 3), and 0.022 ± 0.009 particles/µm² in zone I (Table 3). The fraction of septa containing particles of 1.0-µm diameter decreased from 0.45 ± 0.12 in zone II to 0.27 ± 0.12 at the zone I/II border and to 0.13 ± 0.04 in zone I. Thus both the particle densities and the fraction of perfused septa rose as Ppa was raised from below PA (zone I) to PA (zone I/II border) and above PA, suggesting that the functional diameter of the septal microvessels rose.

This conclusion is also supported by data of the 2.0- and 4.0-µm-diameter particles used in the zone II and zone I/II border studies. For the 2-µm-diameter particles, particle densities within septa increased from 0.019 ± 0.008 particles/µm² at the zone I/II border to 0.032 ± 0.013 particles/µm² in zone II. The fraction of septa perfused with these particles increased from 0.16 ± 0.04 to 0.31 ± 0.12, respectively. A similar trend existed for the 4.0-µm-diameter particles. Particle densities increased from 0.007 ± 0.004 particles/µm² at the zone I/II border to 0.015 ± 0.007 particles/µm² in zone II. The fraction of septa perfused with 4.0-µm-diameter particles increased from 0.09 ± 0.03 to 0.25 ± 0.09, respectively. Thus the trend was toward higher densities within septa and greater numbers of perfused septa as PA was raised from below to above inflation pressure.

In our previous studies (2-4), we estimated functional diameters of septal microvessels by fitting curves produced by the Verniory equation to the particle density data. The Verniory equation was originally developed to estimate diameters of transendothelial pores, based on interstitium-to-plasma concentration ratios for solutes of specific molecular radii (12). We hypothesized that exclusion of latex particles by narrowed septal microvessels could be likened to the reflection of plasma solutes by small transendothelial pores. Using this technique, we estimated diameters of septal microvessels to be 1.7 µm in zone I and 6-8 µm at the zone I/II border. In these previous studies, we did not measure venous effluent particle concentrations but assumed that particles that did not enter septa were reflected away into corner vessels, much as large plasma solutes were reflected by small endothelial pores.

The venous concentration measurements in our present study show that this analogy is inaccurate. Particles that could not enter septal vessels, such as the 5.0- and 6.0-µm-diameter particles, had venous concentrations near zero, indicating that they could not flow through the lung. Thus they were not reflected away from the septal microvessels but were trapped within adjacent, larger vessels.

This finding means that the septal diameter estimates of our previous studies should be revised. In zone I, on the basis of Verniory curves, we estimated the functional diameter of septal microvessels to be 1.7 µm. However, we found that 1.0-µm-diameter particles had very low particle densities within septa (0.022 ± 0.009 particles/µm², Table 3) and found that 4.0-µm-diameter particles were excluded from septa. We did not perfuse with 2.0-µm-diameter particles, which were not available at that time. Based solely on the particle density data, the 1.7-µm-diameter estimate seems reasonable.

At the zone I/II border, on the basis of Verniory curves, we estimated the septal microvessel diameter to be 6-8 µm. However, under these conditions, we found that 4.0-µm-diameter particles had very low particle densities (0.007 ± 0.004 particles/µm², Table 3). We did not perfuse with 5.0-µm-diameter particles, but, on the basis of the low particle density of the 4.0-µm-diameter particles and the fact that 5.0-µm-diameter particles were excluded from septa in zone II (Table 2), it seems likely that these larger particles would have been excluded from septa at the zone I/II border. A more likely microvessel diameter, based solely on the density data, would be near but slightly larger than 4.0 µm.

This is similar to our estimate of the microvessel diameters in zone II. However, we found that the densities of 4.0-µm-diameter particles in zone II were greater than those at the zone I/II border (0.015 ± 0.007 vs. 0.007 ± 0.004 particles/µm²; P  0.05). This suggests that the microvessel diameter was larger in zone II, but the difference must be <1.0 µm, because 5.0-µm-diameter particles were excluded from septa in zone II. Thus the septal microvessel diameters must be 4.0 µm < zone I/II border < zone II < 5.0 µm. This conclusion is supported by our finding that 25 ± 9% of alveoli contained 4.0-µm-diameter particles in zone II (Table 2), whereas only 9 ± 3% of alveoli contained these particles at the zone I/II border (Table 3).

Lamm and colleagues (10), describing red blood cell flow through septa at the pleural surface of intact dog lungs in zone I, reported that red blood cells could be seen flowing through corner vessels but not septal vessels. They reported that, as the lung was placed into zone II, red blood cell flow through septal vessels began. Based on our diameter estimates for these vessels and considering the fact that our studies were not conducted in dogs, the results of Lamm and colleagues suggest that entry of red blood cells into septa in zone II may require some red blood cell deformation. If red blood cells have an undeformed diameter of 5.0 µm and if the septal microvessel diameter is <5.0 µm, as our results suggest, then some red blood cell deformation would be necessary for entry into septa in zone II.

Our septal microvessel diameter estimates are similar to those of others. Guntheroth et al. (7) measured alveolar microvessel diameters from scanning-electron micrographs of latex casts made from rat lungs perfused in zone III. They estimated the microvessel diameter to be 5.8 µm. Glazier and colleagues (6) estimated the diameters to be 2.0-2.8 µm near the zone I/II border. In zone II, they reported that microvessel diameters ranged from 2.8-5.0 µm from the top of the lung to the bottom. Weibel (13) and Pump (11) reported diameters of 6.0 µm for fixed human lungs.

On the other hand, our results are markedly different from those of Fung and colleagues (5), who investigated the patency of pulmonary veins under conditions in which inflation pressure exceeded venous pressure. Their findings are relevant to our present study, because we also set inflation pressure above venous pressure. These investigators filled the vasculature of cat lungs with casting resin and allowed the resin to polymerize while the inflation pressure was set at 10 cmH₂O; the vascular pressure was set 2-17 cmH₂O lower than the inflation pressure (5). Arterial and venous pressures were equal. Examination of the casts revealed that capillary segments were absent. They concluded that, with inflation pressure set above vascular pressure, the capillaries must have been compressed to the point that the resin was excluded. They also found that the smallest pulmonary venous segments in the casts had diameters of 27.4 ± 10.1 µm when vascular pressure was 2 cmH₂O lower than inflation pressure and 23.6 ± 9.4 µm when vascular pressure was 17 cmH₂O lower than inflation pressure. They concluded that the diameters of these smallest patent segments represented choke points in the venous circulation, upstream from which smaller veins and capillaries were collapsed. They concluded that these choke points were responsible for flow limitation in zone II and suggested that the collapsible segments operated in an all-or-none fashion, remaining collapsed until upstream pressure rose enough to open them, then collapsing again as the pressure was relieved.

Our results suggest that flow limitation in zone II occurs because capillaries are narrowed but not collapsed. However, our experiments were conducted under different conditions: we set inflation pressure 15 cmH₂O higher than did Fung and colleagues (5) and allowed the lungs to perfuse continuously. At the high inflation pressures we used, capillaries may be stretched and less collapsible than at the lower pressures used by Fung et al. They suggested that pulmonary veins with diameters larger than the choke point remained open because of tethering by surrounding alveoli. However, at high lung volumes, vascular tension may be great enough to maintain patency of all vessels, including septal microvessels.

These differences between our results and those of Fung and colleagues (5) emphasize the point that zone II is not a specific set of inflation and perfusion pressures but is any condition in which inflation pressure lies between arterial and venous pressures. Septal microvessel diameters in zone II at high lung volumes may be different from those at low or intermediate volumes, which is a possibility that deserves to be addressed by future studies.

A disadvantage of our analytic approach is that we were unable to view particle motion within capillaries as the lung was perfused. Therefore, we could not evaluate the dynamic properties of capillary particle deposition. However, some aspects of these properties can be inferred from the figures. In Fig. 2A, an interseptal corner vessel filled with 4-µm-diameter particles is oriented diagonally across the view. Few of the alveoli supplied by it contain particles, suggesting that few of their septal capillaries were large enough to admit these particles. The implication is that the majority of capillary entrance diameters under these conditions was <4 µm. This would explain why we found that only 25% of the examined alveoli contained particles of this size (Table 2). This suggests that capillary diameters are not uniform; only the largest ones may have been able to admit 4-µm-diameter particles.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Three-dimensional reconstructions of optical sections from a lung perfused with 4-µm-diameter fluorescent latex particles. A: in low-power view, particles (arrows) appear to be confined to a corner vessel pathway that lies between adjacent alveoli. Particles have entered septa of one alveolus (*), which is shown enlarged in B. A total of 49 optical sections were assembled digitally to produce A; 109 sections were assembled to produce B (Voxblast, version 1.0). One section appears in Fig. 1A. Reconstructions are displayed obliquely (right upper corner tipped back) to enhance 3-dimensional effect. Scale is 75 µm (A) and 25 µm (B).

Within the capillary network of individual septa, it has been shown that flow can follow nonrandom pathways through the multiple segments that make up the network (9). Subtle differences in pressure distribution within the network may be responsible for this phenomenon. Similarly, subtle pressure differences at the network entrance might determine whether or not 4-µm-diameter particles can enter. We did not raise Ppa above 30 cmH₂O, but, if we had, more septa might have contained these particles. We also did not ventilate the lungs. Varying the difference between inflation and vascular pressures while maintaining the lung in zone II might also have caused 4-µm-diameter particles to enter more septa.

Our point is that subtle pressure differences might affect the number of septa that particles can enter. Furthermore, capillary opening pressures and functional diameters undoubtedly vary among septa.

We conclude that the largest functional diameter of alveolar septal microvessels at high lung volumes in zone II lies between 4.0 and 5.0 µm. This is similar to our revised estimated diameter for these vessels at the zone I/II border, although the functional diameter in zone II appears to be larger by <1.0 µm. These estimates apply only to the specific zone II pressure conditions under which the lungs were perfused.