Pulmonary arterial morphometry from microfocal X-ray computed tomography.

The objective of this study was to develop an X-ray computed tomographic method for pulmonary arterial morphometry. The lungs were removed from a rat, and the pulmonary arterial tree was filled with perfluorooctyl bromide to enhance X-ray absorbance. At each of four pulmonary arterial pressures (30, 21, 12, and 5.4 mmHg), the lungs were rotated within the cone of the X-ray beam that was projected from a microfocal X-ray source onto an image intensifier, and 360 images were obtained at 1 degrees increments. The three-dimensional image volumes were reconstructed with isotropic resolution with the use of a cone beam reconstruction algorithm. The luminal diameter and distance from the inlet artery were measured for the main trunk, its immediate branches, and several minor trunks. These data revealed a self-consistent tree structure wherein the portion of the tree downstream from any vessel of a given diameter has a similar structure. Self-consistency allows the entire tree structure to be characterized by measuring the dimensions of only the vessels comprising the main trunk of the tree and its immediate branches. An approach for taking advantage of this property to parameterize the morphometry and distensibility of the pulmonary arterial tree is developed.

Pulmonary arterial dilation by inhaled NO: arterial diameter, NO concentration relationship.

The objective of this study was to determine the nitric oxide (NO) concentration and vessel diameter dependence of the pulmonary arterial dilation induced by inhaled NO. Isolated dog lung lobes were situated between a microfocal X-ray source and X-ray detector and perfused with either blood or plasma. Boluses of radiopaque contrast medium were injected into the lobar artery under control conditions, when the pulmonary arteries were constricted by infusion of serotonin and when the serotonin infusion was accompanied by inhalation of from 30 to 960 parts/million NO. Arterial diameter measurements were obtained from X-ray images of vessels having control diameters in the 300- to 3,400-microm range. Serotonin constricted the vessels throughout the size range studied, with an average decrease in diameter of approximately 20%. The fractional reversal of the serotonin-induced constriction by inhaled NO was directly proportional to inhaled NO concentration, inversely proportional to vessel size, and greater with plasma than with blood perfusion in vessels as large as 3 mm in diameter. The latter indicates that intravascular hemoglobin affected the bronchoalveolar-to-arterial luminal NO concentration gradient in fairly large pulmonary arteries. The data provide information regarding pulmonary arterial smooth muscle accessibility to intrapulmonary gas that should be useful as part of the database for modeling the communication between intrapulmonary gas and pulmonary arterial smooth muscle cells in future studies.

Microfocal X-ray CT imaging and pulmonary arterial distensibility in excised rat lungs.

The objective of this study was to develop an X-ray computed tomographic method for measuring pulmonary arterial dimensions and locations within the intact rat lung. Lungs were removed from rats and their pulmonary arterial trees were filled with perfluorooctyl bromide to enhance X-ray absorbance. The lungs were rotated within the cone of the X-ray beam projected from a microfocal X-ray source onto an image intensifier, and 360 images were obtained at 1 degrees increments. The three-dimensional image volumes were reconstructed with isotropic resolution using a cone beam reconstruction algorithm. The vessel diameters were obtained by fitting a functional form to the image of the vessel circular cross section. The functional form was chosen to take into account the point spread function of the image acquisition and reconstruction system. The diameter measurements obtained over a range of vascular pressures were used to characterize the distensibility of the rat pulmonary arteries. The distensibility coefficient alpha [defined by D(P) = D(0)(1 + alphaP), where D(P) is the diameter at intravascular pressure (P)] was approximately 2.8% mmHg and independent of vessel diameter in the diameter range (about 100 to 2,000 mm) studied.

Structure-function relationships in the pulmonary arterial tree.

Knowledge of the relationship between structure and function of the normal pulmonary arterial tree is necessary for understanding normal pulmonary hemodynamics and the functional consequences of the vascular remodeling that accompanies pulmonary vascular diseases. In an effort to provide a means for relating the measurable vascular geometry and vessel mechanics data to the mean pressure-flow relationship and longitudinal pressure profile, we present a mathematical model of the pulmonary arterial tree. The model is based on the observation that the normal pulmonary arterial tree is a bifurcating tree in which the parent-to-daughter diameter ratios at a bifurcation and vessel distensibility are independent of vessel diameter, and although the actual arterial tree is quite heterogeneous, the diameter of each route, through which the blood flows, tapers from the arterial inlet to essentially the same terminal arteriolar diameter. In the model the average route is represented as a tapered tube through which the blood flow decreases with distance from the inlet because of the diversion of flow at the many bifurcations along the route. The taper and flow diversion are expressed in terms of morphometric parameters obtained using various methods for summarizing morphometric data. To help put the model parameter values in perspective, we applied one such method to morphometric data obtained from perfused dog lungs. Model simulations demonstrate the sensitivity of model pressure-flow relationships to variations in the morphometric parameters. Comparisons of simulations with experimental data also raise questions as to the "hemodynamically" appropriate ways to summarize morphometric data.

Transit time dispersion in the pulmonary arterial tree.

Knowledge of the contributions of arterial and venous transit time dispersion to the pulmonary vascular transit time distribution is important for understanding lung function and for interpreting various kinds of data containing information about pulmonary function. Thus, to determine the dispersion of blood transit times occurring within the pulmonary arterial and venous trees, images of a bolus of contrast medium passing through the vasculature of pump-perfused dog lung lobes were acquired by using an X-ray microfocal angiography system. Time-absorbance curves from the lobar artery and vein and from selected locations within the intrapulmonary arterial tree were measured from the images. Overall dispersion within the lung lobe was determined from the difference in the first and second moments (mean transit time and variance, respectively) of the inlet arterial and outlet venous time-absorbance curves. Moments at selected locations within the arterial tree were also calculated and compared with those of the lobar artery curve. Transit times for the arterial pathways upstream from the smallest measured arteries (200-micron diameter) were less than approximately 20% of the total lung lobe mean transit time. Transit time variance among these arterial pathways (interpathway dispersion) was less than approximately 5% of the total variance imparted on the bolus as it passed through the lung lobe. On average, the dispersion that occurred along a given pathway (intrapathway dispersion) was negligible. Similar results were obtained for the venous tree. Taken together, the results suggest that most of the variation in transit time in the intrapulmonary vasculature occurs within the pulmonary capillary bed rather than in conducting arteries or veins.

Anatomic distribution of pulmonary vascular compliance.

Previously, the pressure changes after arterial and venous occlusion have been used to characterize the longitudinal distribution of pulmonary vascular resistance with respect to vascular compliance using compartmental models. However, the compartments have not been defined anatomically. Using video microscopy of the subpleural microcirculation, we have measured the flow changes in approximately 40-micron arterioles and venules after venous, arterial, and double occlusion maneuvers. The quasi-steady flows through these vessels after venous occlusion permitted an estimation of the compliance in three anatomic segments: arteries > 40 microns, veins > 40 microns, and vessels < 40 microns in diameter. We found that approximately 65% of the total pulmonary vascular compliance was in vessels < 40 microns, presumably mostly capillaries. The transient portions of the pressure and flow data after venous, arterial, and double occlusion were consistent with most of the arterial compliance being upstream from most of the arterial resistance and most of the venous compliance being downstream from most of the venous resistance.

Flow-induced vasodilation in the ferret lung.

To examine the possibility that shear stress may be a pulmonary vasodilator stimulus, we studied the effect of changing blood flow on the diameters of small pulmonary arteries in isolated perfused ferret lung lobes. The arteries studied were in the approximately 0.3- to 1.3-mm-diameter range, and the diameters were measured by using microfocal X-ray imaging. The diameters were measured at two flow rates, 10 and 40 ml/min, with the intravascular pressure in the measured vessels the same at the two flow rates as the result of venous pressure adjustment. The response to a change in flow was studied under both normoxic and hypoxic conditions. Hypoxia was used to elevate pulmonary arterial tone to increase the likelihood of detecting a vasodilator response. Under normoxic conditions, changing flow had little effect on the arterial diameters, but under hypoxic conditions the arteries were consistently larger at the higher flow than at the lower flow, even though the distending pressure was the same at the two flow rates. The results are consistent with the hypothesis that shear stress is a pulmonary vasodilator stimulus.

Hypoxic vasoconstriction in pulmonary arterioles and venules.

Pulmonary microvessels (<70 microm) lack a complete muscular media. We tested the hypothesis that these thin-walled vessels do not participate in the hypoxic pressor response. Isolated canine lobes were pump perfused at precisely known microvascular pressures. A videomicroscope, coupled to a computerized image-enhancement system, permitted accurate diameter measurements of subpleural arterioles and venules, with each vessel serving as its own control. While vascular pressure was maintained constant throughout the protocol, hypoxia caused an average reduction of 25% of microvessel diameters. The constriction was reversed when nitric oxide was added to the hypoxic gas mixture. The nitric oxide reversal, combined with a lack of lobar blood flow redistribution as measured by fluorescent microspheres, shows that the constriction was active. This response suggests the unexpected potential for active intra-acinar ventilation-perfusion matching.

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.

Distribution of pulmonary capillary red blood cell transit times.

In theory, red blood cells can pass through the pulmonary capillaries too rapidly to be completely saturated with oxygen during exercise. This idea has not been directly tested because the transit times of the fastest red blood cells are unknown. We report the first measurements of the entire transit time distribution for red blood cells crossing single subpleural capillary networks of canine lung using in vivo fluorescence videomicroscopy and compare those times with the distribution of plasma transit times in the same capillary networks. On average, plasma took 1.4 times longer than red blood cells to pass through the capillary bed. Decreased transit times with increased cardiac output were mitigated by both capillary recruitment and a narrowing of the transit time distribution. This design feature of the pulmonary capillary bed kept the shortest times from falling below the theoretical minimum time for complete oxygenation.

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.

Measuring pulmonary microvessel diameters using video image analysis.

To directly determine the pressure-diameter relationship of individual pulmonary microvessels, it is necessary to measure the width of the column of blood in the vessel because microvascular walls are invisible when using intravital microscopy. To identify the margins of the blood column accurately, we developed a method for computer enhancement and measurement of vessel images. After recording microvessels on videotape, consecutive frames from the videotape were digitized by a computer. Pixels that changed from frame to frame (moving erythrocytes) were turned white, and unchanging pixels were turned black. In this way an image of the erythrocyte column with distinct edges was produced. The width of this column was measured with a heuristic technique involving interactions between the computer and the user. The measurements were reproducible and accurate. This technique has been used to measure microvascular diameters over a range of well-defined microvascular pressures and construct precise pressure-diameter curves.

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.

Effect of increasing flow on distribution of pulmonary capillary transit times.

The complex morphology of the pulmonary capillary network causes capillary transit times to be dispersed about a mean. It is known that flow-induced decreases in mean capillary transit time are partially offset by capillary recruitment and distension, but the effect of these factors on the rest of the distribution of transit times is unknown. We have studied the relationship between blood flow, capillary recruitment, and the distribution of transit times in isolated canine lungs with videomicroscopy. Doubling baseline lobar blood flow recruited capillaries. All transit times in the distribution decreased, as did relative dispersion. Doubling flow again caused a further decrease in transit times, but neither capillary recruitment nor relative dispersion changed significantly. We conclude that capillary transit times become more homogeneous as lobar flow increases from low to intermediate levels. Further increases in flow across a fully recruited network are associated with decreases in transit times but not with more homogeneous capillary perfusion.

Direct measurement of pulmonary microvascular distensibility.

Pulmonary vascular distensibility has an important influence on pulmonary hemodynamics. Although many measurements of distensibility have been made on large pulmonary vessels, there is less information on microvascular distensibility. We have measured the distensibility of the smallest (< 70-microns-diam) precapillary arterioles and postcapillary venules. Isolated dog lobes, at 2.5 cmH2O transpulmonary pressure, were perfused at low flows, which caused the arteriovenous pressure gradient to be very small and thereby permitted accurate estimation of microvascular pressure. As microvascular pressure was systematically varied between 0 and 30 mmHg, subpleural microvascular diameters were determined from computer-enhanced images obtained by videomicroscopy. Arteriolar and venular distensibilities were not different from each other. The microvascular pressure-diameter relationship was alinear with distensibility coefficients of 1-3% mmHg-1, values that are of the same order of magnitude as previously measured distensibilities of 100- to 1,000-microns-diam canine pulmonary vessels.

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.