Computer determination of perfusion patterns in pulmonary capillary networks.

Individual pulmonary capillaries are not steadily perfused. By using in vivo microscopy, it can readily be demonstrated that perfusion continually switches between capillary segments and between portions of the network within a single alveolar wall. These changes in capillary perfusion occur even when upstream pressure and flow are constant. Flow switching between capillary segments in the absence of hemodynamic changes in large upstream vessels suggests that capillary perfusion patterns could be random. To calculate the probability that perfusion patterns could occur by chance, it is necessary to know the total number of possible perfusion patterns in a given capillary network. We developed a computer program that can determine every possible perfusion pattern for any given capillary network, and from that information we can calculate whether perfusion of individual segments in the network is random. With the results of the computer program, we have obtained statistical evidence that some capillary segments in a network are nonrandomly perfused.

Pulmonary capillary diameters and recruitment characteristics in subpleural and interior networks.

In vivo microscopic observations of pulmonary capillaries are limited to subpleural networks that are less dense than interior networks. In addition to the density difference, subpleural and interior capillary diameters may differ, although there are conflicting data on this point. We measured the diameters of subpleural and interior capillaries in rats and dogs. Subpleural diameters were 30% larger in rats and 20% larger in dogs. Because diameter and density differences might cause differences in recruitment between subpleural and interior networks, we measured subpleural and interior recruitment by counting the number of red blood cells per 10 microns of alveolar wall in histological cross sections of rapidly frozen rat lungs. Lung inflation pressures of 4, 12, and 25 cmH2O created a wide range of capillary recruitment in different groups of animals. Red blood cell counts for interior and subpleural capillaries moved in parallel and progressively increased as inflation pressures were reduced. These data demonstrate that recruitment in subpleural capillaries accurately reflect recruitment in interior capillaries and validate the use of in vivo microscopic observations of subpleural capillaries to investigate pulmonary capillary recruitment in general.

Sites of leukocyte sequestration in the pulmonary microcirculation.

The location and mechanisms of leukocyte sequestration in the pulmonary circulation have been investigated by using high-magnification in vivo videomicroscopy to record the passage of unlabeled native leukocytes through canine pulmonary capillaries. Of 650 leukocytes traversing capillary networks, 46 +/- 6% (SE) of the leukocytes passed through without stopping, 42 +/- 9% stopped in segments between junctions, and 12 +/- 4% stopped in junctions. Leukocytes rolling along arteriolar walls were nearly spherical, as 94% had aspect ratios (major axis divided by minor axis) < or = 1.25. To pass through the capillary bed, the leukocytes deformed into elongated shapes. Many leukocytes remained elongated after entering the venules (53% had aspect ratios > or = 1.25). Venular rolling was blocked by fucoidin (blocking both L- and P-selectin) but not by anti-P-selectin antibodies alone, indicating that rolling leukocytes adhered to the venular endothelium by L-selectin. These observations demonstrate that leukocytes deform to transit the capillary bed, that they stop more frequently in segments than in junctions, and that rolling leukocytes in the venular marginated pool adhere via L-selectin.

Stability of alveolar capillary opening pressures.

Little is known about the stability of the process by which pulmonary capillaries open. To investigate this process, pulmonary capillary perfusion patterns in isolated pump-perfused canine lobes were studied using video microscopy. After pump flow was set to perfuse one-half of the capillaries, the pump was turned off and all of the capillaries emptied. Turning the pump back on reopened the capillaries. The on-off cycle was repeated six times. If the same capillaries were perfused during each observation, it would demonstrate that there were stable and significant differences between individual capillary opening pressures, causing consistent recruitment of those capillaries with the lowest opening pressures. Alternatively, variable perfusion patterns would result if capillary opening pressures changed between observations, if the differences in opening pressures between capillary segments were negligible, or if experimental conditions changed between cycles. The perfusion pattern was more reproducible than expected by chance alone, which indicated the existence of stable differences among alveolar capillary opening pressures.

Temporal capillary perfusion patterns in single alveolar walls of intact dogs.

Pulmonary gas exchange reserve in the form of recruitable capillaries was first described in the 1930s, when in vivo microscopy was used to demonstrate that not all capillaries were perfused during basal conditions and that perfusion of individual capillaries varied over time. These important observations have never been directly confirmed, nor have the hemodynamic causes of the variation been investigated. We used videomicroscopy to record nine consecutive pulmonary capillary perfusion patterns during a 40-min period. Confirming the original work, we found considerable perfusion variation in about one-half of the capillaries. These variations did not correlate with changes in pulmonary arterial pressures or cardiac outputs, suggesting that factors more subtle than large-vessel hemodynamics affected capillary perfusion consistency. In contrast to this variable group, one-half of the capillary segments were consistently perfused during at least eight of the nine observations and were interconnected to form preferential pathways across the alveolar wall.

Computer simulation of neutrophil transit through the pulmonary capillary bed.

One-half of the neutrophils that enter the pulmonary circulation become temporarily trapped in capillaries. The neutrophils that are impeded make complete stops between free-flowing movements. These observations, based on in vivo microscopy, suggest that pulmonary margination is caused by neutrophils being impeded at focal sites in the capillary bed. To investigate the frequency with which impeding sites had to occur in the pulmonary capillaries to trap one-half of the circulating neutrophils, we developed a computer model to simulate neutrophils encountering discrete obstructions in a capillary-like network. Surprisingly, if only 1% of the capillaries in the network acted as traps, one-half of the neutrophils stopped at least once. The trapping ability of a given percentage of obstructions was independent both of the geometry of the network was whether the obstructions occurred in the segments or junctions. To simulate neutrophil transit more realistically, both neutrophil and capillary diameters were randomly selected from published diameter distributions. Every neutrophil was trapped multiple times by this model, suggesting that cell deformation contributes importantly to neutrophil passage through the pulmonary capillary bed.

Capillary perfusion patterns in single alveolar walls.

We studied capillary perfusion patterns in single alveolar walls through a transparent thoracic window implanted in pentobarbital-anesthetized dogs. The capillaries were maximally opened by brief inflation of a balloon in the left atrium to raise pressure. After the balloon was deflated and pulmonary hemodynamics returned to zone 2 baseline conditions, the capillaries that remained perfused in the observed field were videotaped with the use of in vivo microscopy. The cycle of elevated pressure and baseline observation was repeated three times. Perfusion of different capillaries during each of the observations would imply that the capillaries had characteristics that permitted flow to switch between segments. Perfusion of a specific set of pathways through the network each time would demonstrate that flowing blood sought a unique and repeatable combination of segments, presumably with the least total pathway resistance. We found that the same capillary segments were perfused 79% of the time, a strong indication that a reproducible combination of individual segmental resistances determined the predominant pattern of pulmonary capillary perfusion.

Neutrophil kinetics in the pulmonary microcirculation during acute inflammation.

The site of neutrophil interaction with the vasculature during acute lung inflammation is controversial, but has been suggested to occur in the alveolar capillaries, in contrast with its location in postcapillary venules in nonpulmonary tissues. We studied the kinetics of neutrophil accumulation and the site of neutrophil-vascular interaction in the lung by examining directly the behavior of fluorescein isothiocyanate-labeled canine neutrophils utilizing in vivo fluorescence videomicroscopy through a window inserted into the chest wall of anesthetized dogs. The administration of fragments of the fifth component of complement (C5f) into either the airway or pulmonary artery resulted in neutrophil sequestration almost exclusively in pulmonary capillaries. Kinetically, there was a shift in the distribution of neutrophil transit times resulting in a marked prolongation of median transit time. This response occurred within seconds after intravascular C5f and within 5 minutes after airway C5f and was maintained for at least 30 minutes. Ultrastructural studies after airway C5f showed neutrophils in various stages of migration through the alveolar-capillary membrane and more than 90% of these neutrophils were seen to migrate from capillary rather than from venular sites. These data indicate that pulmonary inflammation differs from inflammation in other vascular beds primarily in the site of neutrophil localization and migration. This fundamental difference in the inflammatory response may serve to localize the inflammatory response to the alveolus, and (since cells were retained singly), indicates the inability of leukoaggregation adequately to explain the findings. Leukocyte accumulation in the lung may thus occur through alterations in the balance between delivery of neutrophils to the lung and the transit time of these cells across the capillary bed.

Distribution of pulmonary capillary transit times in recruited networks.

When pulmonary blood flow is elevated, hypoxemia can occur in the fastest-moving erythrocytes if their transit times through the capillaries fall below the minimum time for complete oxygenation. This desaturation is more likely to occur if the distribution of capillary transit times about the mean is large. Increasing cardiac output is known to decrease mean pulmonary capillary transit time, but the effect on the distribution of transit times has not been reported. We measured the mean and variance of transit times in single pulmonary capillary networks in the dependent lung of anesthetized dogs by in vivo videofluorescence microscopy of a fluorescein dye bolus passing from an arteriole to a venule. When cardiac output increased from 2.9 to 9.9 l/min, mean capillary transit time decreased from 2.0 to 0.8 s. Because transit time variance decreased proportionately (relative dispersion remained constant), increasing cardiac output did not alter the heterogeneity of local capillary transit times in the lower lung where the capillary bed was nearly fully recruited.

Pulmonary microcirculatory kinetics of neutrophils deficient in leukocyte adhesion-promoting glycoproteins.

The mechanism that causes neutrophils to sequester in the pulmonary circulation is unknown. Because the CD11/CD18 glycoprotein family on the surface membrane of neutrophils participates in many adhesive interactions with the endothelium, we investigated the role of these proteins in the intravascular sequestration of pulmonary neutrophils. Neutrophils were isolated from normal dogs and from the only living dog known to have leukocyte adhesion deficiency disease, an inherited deficiency of the CD11/CD18 adhesion family. The neutrophils were labeled with fluorescein dye, injected into normal recipient dogs, and their passage through the pulmonary microcirculation was recorded by in vivo videofluorescence microscopy through a transparent thoracic window. Transit times for normal and deficient neutrophils were similar over a wide range of hemo-dynamic conditions. Activation by zymosan-activated plasma, which increases the surface membrane expression of CD11/CD18, prolonged the transit of normal neutrophils but did not alter the transit time of the deficient neutrophils. These results indicate that neutrophil CD11/CD18 adhesion-promoting glycoproteins are not involved in the normal pulmonary sequestration of neutrophils but have a significant role in the arrest of activated neutrophils in the pulmonary capillaries.

Neutrophil kinetics in the pulmonary microcirculation. Effects of pressure and flow in the dependent lung.

Increases in pulmonary arterial pressure or blood flow raise peripheral white cell count by releasing sequestered leukocytes from the lung. The effects of altered hemodynamics, however, on the leukocyte sequestration site and on the distribution of leukocyte transit times through the pulmonary microcirculation are unknown. We used in vivo fluorescence videomicroscopy to study the passage of individual, fluorescein-isothiocyanate-labeled neutrophils through the pulmonary microcirculation of anesthetized dogs. Pulmonary hemodynamics were altered over a wide range. Regardless of the hemodynamic conditions, the only place that any of the 2,919 observed neutrophils stopped was in the capillaries. The periods of immobility had a wide range, from less than 1 to greater than 1,200 s. Because the cells remained motionless once they stopped and then accelerated suddenly as they regained the free-flowing stream, the obstructions must have been discrete. About a quarter of the capillary pathways had one site of high resistance. Another quarter offered two or more obstructions. In the remaining half, the neutrophils passed rapidly and without pause from arteriole to venule. Increases in pressure and flow decreased the number of times that individual cells stopped. These changes altered the median transit time by shifting the distribution of transit times between the slowest and fastest groups. We conclude that most of the total pathlength of perfused capillaries offers little resistance even to neutrophils. There are, however, focal areas in individual capillaries that offer high resistance to neutrophil passage.

Site of recruitment in the pulmonary microcirculation.

Increasing the total surface area of the pulmonary blood-gas interface by capillary recruitment is an important factor in maintaining adequate oxygenation when metabolic demands increase. Capillaries are known to be recruited during conditions that raise pulmonary blood flow and pressure. To determine whether pulmonary arterioles and venules are part of the recruitment process, we made in vivo microscopic observations of the subpleural microcirculation (all vessels less than 100 microns) in the upper lung where blood flow is low (zone 2). To evoke recruitment, pulmonary arterial pressure was elevated either by an intravascular fluid load or by airway hypoxia. Of 209 arteriolar segments compared during low and high pulmonary arterial pressures, none recruited or derecruited. Elevated arterial pressure, however, did increase the number of perfused capillary segments by 96% with hypoxia and 165% with fluid load. Recruitment was essentially absent in venules (4 cases of recruitment in 289 segments as pressure was raised). These data support the concept that recruitment in the pulmonary circulation is exclusively a capillary event.

Pulmonary arterial transit times.

To begin to characterize the pulmonary arterial transport function we rapidly injected a bolus containing a radiopaque dye and a fluorescence dye into the right atrium of anesthetized dogs. The concentrations of the dye indicators were measured in the main pulmonary artery (fluoroscopically) and in a subpleural pulmonary arteriole (by fluorescence microscopy). The resulting concentration vs. time curves were subjected to numerical deconvolution and moment analysis to determine how the bolus was dispersed as it traveled through the arteriole stream tube from the main pulmonary artery to the arteriole. The mean transit time and standard deviation of the transport function from the main pulmonary artery to the arterioles studied averaged 1.94 and 1.23 s, respectively, and the relative dispersion (ratio of standard deviation to mean transit time) was approximately 64%. This relative dispersion is at least as large as those reported for the whole dog lung, indicating that relative to their respective mean transit times the dispersion upstream from the arterioles is comparable to that taking place in capillaries and/or veins. The standard deviations of the transport functions were proportional to their mean transit times. Thus the relative dispersion from the main pulmonary artery to the various arterioles studied was fairly consistent. However, there were variations in mean transit time even between closely adjacent arterioles, suggesting that variations in mean transit times between arteriole stream tubes also contribute to the dispersion in the pulmonary arterial tree.

Comparison of direct and indirect measurements of pulmonary capillary transit times.

Using in vivo microscopy, we made direct measurements of pulmonary capillary transit time by determining the time required for fluorescent dye to pass from an arteriole to a venule on the dependent surface of the dog lung. Concurrently, in the same animals, pulmonary capillary transit time was measured indirectly in the entire lung using the diffusing capacity method (capillary blood volume divided by cardiac output). Transit times by each method were the same in a group of five dogs [direct: 1.75 +/- 0.27 (SE) s; indirect: 1.85 +/- 0.33 s; P = 0.7]. The similarity of these transit times is important, because the widely used indirect determinations based on diffusing capacity are now shown to coincide with direct measurements and also because it demonstrates that measurements of capillary transit times on the surface of the dependent lung bear a useful relationship to measurements on the capillaries in the rest of the lung.

Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries.

Although the lung is known to be a major site of neutrophil margination, the anatomic location of these sequestered cells within the lung is controversial. To determine the site of margination and the kinetics of neutrophil transit through the pulmonary microvasculature, we infused fluorescein isothiocyanate-labeled canine neutrophils into the pulmonary arteries of 10 anesthetized normal dogs and made fluorescence videomicroscopic observations of the subpleural pulmonary microcirculation through a window inserted into the chest wall. The site of fluorescent neutrophil sequestration was exclusively in the pulmonary capillaries with a total of 951 labeled cells impeded in the capillary bed for a minimum of 2 s. No cells were delayed in the arterioles or venules. Transit times of individual neutrophils varied over a wide range from less than 2 s to greater than 20 min with an exponential distribution skewed toward rapid transit times. These observations indicate that neutrophil margination occurs in the pulmonary capillaries with neutrophils impeded for variable periods of time on each pass through the lung. The resulting wide distribution of transit times may determine the dynamic equilibrium between circulating and marginated neutrophils.

Vertical gradient of pulmonary capillary transit times.

The key determinants of alveolar capillary perfusion are transit times and the extent of recruitment. Capillaries are known to be heavily recruited in the dependent lung, but there are no direct data that bear on how capillary transit times might be affected by gravity. We directly determined mean capillary transit times on the surface of the upper, middle, and lower lung by measuring the passage of fluorescent dye through the capillaries using in vivo television microscopy. In anesthetized dogs, mean capillary transit times averaged 12.3 s in the upper lung, 3.1 s in the midlung, and 1.6 s in the lower lung. This near order of magnitude variation in speed of blood transit establishes that there is a vertical gradient of capillary transit times in the lung. As expected, dependent capillary networks were nearly fully recruited, whereas relatively few capillaries were perfused in the upper lung. The lengthy transit times and sparsely perfused capillary beds in the upper lung combine to provide a substantial part of pulmonary gas exchange reserve.

Direct measurement of pulmonary capillary transit times.

Direct measurements of capillary transit times in dog lungs were made by using in vivo television microscopy. Mean transit times were unexpectedly long. As pulmonary artery pressures were raised, transit times decreased, suggesting that the normally existing hydrostatic pressure gradient in the lung causes a vertical distribution of transit times. The more rapid transit times approached the minimum time required for complete oxygenation.

Intrapulmonary blood flow redistribution during hypoxia increases gas exchange surface area.

We have previously shown that airway hypoxia causes pulmonary capillary recruitment and raises diffusing capacity for carbon monoxide. This study was designed to determine whether these events were caused by an increase in pulmonary vascular resistance, which redistributed blood flow toward the top of the lung, or by an increase in cardiac output. We measured capillary recruitment at the top of the dog lung by in vivo microscopy, gas exchange surface area of the whole lung by diffusing capacity for carbon monoxide, and blood flow distribution by radioactive microspheres. During airway hypoxia recruitment occurred, diffusing capacity increased, and blood flow was redistributed upward. When a vasodilator was infused while holding hypoxia constant, these effects were reversed; i. e., capillary "derecruitment" occurred, diffusing capacity decreased, and blood flow was redistributed back toward the bottom of the lung. The vasodilator was infused at a rate that left hypoxic cardiac output unchanged. These data show that widespread capillary recruitment during hypoxia is caused by increased vascular resistance and the resulting upward blood flow redistribution.

Diffusing capacity of the lung during hypoxia: role of capillary recruitment.

We have used in vivo microscopy to show that airway hypoxia caused pulmonary capillary recruitment. To determine whether the recruited capillaries add to the surface area for gas exchange, we measured the diffusing capacity of the lung for carbon monoxide before and after inducing recruitment with hypoxia. Diffusing capacity increased during hypoxia. However, some increase in diffusing capacity was expected, since there were fewer oxygen molecules to compete with carbon monoxide for hemoglobin-binding sites. To determine the effect of capillary recruitment alone on diffusing capacity, we held hypoxia constant while infusing a vasodilator to diminish recruitment. Diffusing capacity decreased concomitantly as recruitment diminished. These results indicate that our microscopic observations reflect a widespread recruitment that increases the gas exchange surface area.

Capillary recruitment during airway hypoxia: role of pulmonary artery pressure.

Hypoxia has been shown to cause an increased number of pulmonary capillaries to be perfused. Changes in cardiac output and left atrial pressure have been previously ruled out as causes of this capillary recruitment. Increased pulmonary vein pressure and increased pulmonary artery pressure remain as two potential mechanisms. To differentiate between these two possible causes, we measured pulmonary artery and vein pressures with directly placed catheters and capillary recruitment with in vivo microscopy. During isocapnic hypoxia pulmonary artery pressure doubled, observed capillary recruitment increased fivefold, and pulmonary vein pressure remained constant. When the vasodilator prostaglandin E1 was infused during hypoxia, pulmonary artery pressure and capillary recruitment fell to control values and pulmonary vein pressure remained constant. Since capillary recruitment correlated with pulmonary artery pressure in each dog, but not with pulmonary vein pressure, we conclude that arterial, not venous, constriction is the probable cause of this recruitment.