J Appl Physiol 102: 1265-1272, 2007.
First published December 7, 2006; doi:10.1152/japplphysiol.00756.2006
8750-7587/07 $8.00
INVITED REVIEW
HIGHLIGHTED TOPIC
Physiological Imaging of the Lung
Microsphere maps of regional blood flow and regional ventilation
H. Thomas Robertson and
Michael P. Hlastala
Departments of Medicine and of Physiology and Biophysics, University of Washington, Seattle, Washington
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ABSTRACT
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Systematically mapped samples cut from lungs previously labeled with intravascular and aerosol microspheres can be used to create high-resolution maps of regional perfusion and regional ventilation. With multiple radioactive or fluorescent microsphere labels available, this methodology can compare regional flow responses to different interventions without partial volume effects or registration errors that complicate interpretation of in vivo imaging measurements. Microsphere blood flow maps examined at different levels of spatial resolution have revealed that regional flow heterogeneity increases progressively down to an acinar level of scale. This pattern of scale-dependent heterogeneity is characteristic of a fractal distribution network, and it suggests that the anatomic configuration of the pulmonary vascular tree is the primary determinant of high-resolution regional flow heterogeneity. At 
2-cm3 resolution, the large-scale gravitational gradients of blood flow per unit weight of alveolar tissue account for <5% of the overall flow heterogeneity. Furthermore, regional blood flow per gram of alveolar tissue remains relatively constant with different body positions, gravitational stresses, and exercise. Regional alveolar ventilation is accurately represented by the deposition of inhaled 1.0-µm fluorescent microsphere aerosols, at least down to the 2-cm3 level of scale. Analysis of these ventilation maps has revealed the same scale-dependent property of regional alveolar ventilation heterogeneity, with a strong correlation between ventilation and blood flow maintained at all levels of scale. The ventilation-perfusion (
A/
) distributions obtained from microsphere flow maps of normal animals agree with simultaneously acquired multiple inert-gas elimination technique A/ distributions, but they underestimate gas-exchange impairment in diffuse lung injury.
ventilation-perfusion ratio; radioactive microspheres; fluorescent microspheres; aerosol
IN VIVO PULMONARY IMAGES OF intravenously injected radionuclide-labeled microspheres and radionuclide-labeled aerosol have been part of the clinical diagnostic armamentarium for over 40 years. The resolution of the old in vivo planar gamma camera images was relatively weak, and lung flow mapping techniques using experimental animals were subsequently developed to gain enhanced spatial resolution. The microsphere mapping approach involves in vivo administration of intravascularly injected and/or aerosol labels, followed by fixation of the lung, systematic cutting and mapping of the cut pieces, and measurement of label concentrations in each piece. The spatial perfusion and/or ventilation maps that are reconstructed from these data have been employed in a wide range of experimental studies. This review will address the advantages and limitations of this approach to map regional pulmonary perfusion and regional ventilation, and it will outline the important concepts that have arisen from studies utilizing the technique.
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ADVANTAGES OF MICROSPHERE MAPS
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In comparison to in vivo imaging techniques utilizing radionuclide-labeled microspheres (RMS) to estimate regional blood flow, destructive studies of lungs previously labeled with intravenously injected RMS have three important advantages. First, RMS regional blood flow measurements made on cut pieces of lung are not influenced by partial volume artifact. Hence, provided there is adequate signal from the label, the level of spatial resolution attainable by the destructive approach is simply a function of the cut piece size. Second, for studies of responses to different interventions, with each condition marked by a different injected microsphere label, there is no ambiguity concerning the exact spatial region sampled at the time of each measurement. Finally, because all nonmicrosphere imaging modalities to estimate regional blood flow require a mathematical model for interpretation, the intravascular microsphere deposition method sidesteps the assumptions of those models.
Fluorescent microspheres (FMS) can be employed as flow markers for maps, because the small tissue pieces sampled by this destructive methodology permit complete extraction of fluorescence from either inhaled and deposited aerosol or intravenously injected microspheres. The availability of 13 different fluorescent labels has substantially expanded investigational opportunities for simultaneous measurements of both regional perfusion and regional ventilation with the small tissue samples (51). The correlation for simultaneously administered different FMS colors uniformly exceeds 0.99 for the standard 2-cm3 lung piece size, and these measurements correlate equally well with simultaneous measurements made with RMS (18). Relative to RMS, FMS labels show only a modest signal overlap with combinations of different labels, and as with RMS, this overlap is readily corrected by a matrix inversion program (51). Properly stored FMS maintain their fluorescent intensity over time and also remain stable over extended periods of time when injected into experimental animals (23). Finally, FMS markers are particularly convenient to use in studies of the lung, because the fluorescence can be completely extracted from dried lung pieces by soaking in a solvent, without requiring tissue digestion, as is necessary for FMS measurements in other organs (18).
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LIMITATIONS OF MICROSPHERE MAPS
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An initial concern for any microsphere study of regional blood flow was whether there might be a systematic bias, because 15-µm microspheres are large, unyielding particles relative to the characteristics of erythrocytes. Bassingthwaighte et al. (6) utilized mapped measurements of an atrially injected radionuclide-labeled "molecular microsphere" that was fully extracted by myocardial capillary endothelium and compared those measurements with data obtained by injection of standard 15-µm RMS. They found excellent correlation between the two regional flow measurements, with only a modest tendency for microspheres to underestimate the very lowest flows and overestimate the very highest flows. Using an isolated perfused lung, Beck (7) compared regional microsphere deposition with flow of radionuclide-labeled erythrocytes, and he found no significant difference between measurements. Melsom et al. (43) made a comparison of lung RMS blood flow maps with pulmonary parenchymal extraction of a radionuclide-labeled molecular microsphere and again demonstrated no significant difference between the two measurements of regional flow.
A concern for interpretation of microsphere maps of pulmonary flow is that the lung can only be represented by one volume, ordinarily the total lung capacity (TLC). The in vivo apex-to-base density gradient in an upright lung or the in vivo ventral-to-dorsal density gradient in a supine lung is not appropriately represented when the lung is extracted and air-dried inflated to TLC. In the prone position, lung parenchymal density is relatively uniform (33), and the most appropriate comparisons between microsphere maps and in vivo measurements should be made using in vivo measurements acquired in the prone position.
Multiple lung samples are required to construct microsphere maps, and each of the two generally used cutting options has pros and cons. If the entire organ is sampled by cutting along a rectilinear grid, then the entire cardiac output and alveolar ventilation during labeling are represented but with the disadvantage that the rectilinear sectioning produces a large fraction of irregularly sized pieces from the periphery of the lung. Without adjustment, the lower signal obtained from the smaller edge pieces creates the appearance of a radial gradient of flow. The correction employed in most studies is to normalize the microsphere signal by the weight of the air-dried piece that has been flushed clear of blood, the so-called "flow per alveolus" correction. A problem with this convention is that it underestimates the regional flow for lung pieces that include the relatively heavy large airways, or regions with inflammation or scarring. Another issue with the flow per alveolus correction is that for animals such as sheep and goats, with lungs heavily invested with connective tissue, alveolar tissue content is less well represented by tissue weight. An alternative to weight normalization is to systematically sample a subset of fixed-volume pieces, avoiding the most peripheral parts of lung, large conducting airways, and large vasculature. This approach has the advantage of appropriately characterizing large-scale spatial gradients with fewer samples, and the uniform piece size provides an estimate of regional flow heterogeneity without the bias introduced by giving identical statistical weight to tissue samples of different volumes. The disadvantage is that small-scale heterogeneity is less well characterized, and influences of the most peripheral and most central parts of the lung are ignored.
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METHODS TO CREATE MICROSPHERE MAPS OF FLOW
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The initial studies utilizing measurements of intravenously injected RMS to reconstruct microsphere flow maps of the lung employed different approaches to lung fixation, sectioning, flow measurement, and data reduction. Regardless of the method, these studies revealed an unexpected extent of flow heterogeneity that was initially attributed to problems with the methods themselves. Hogg et al. (34) compared distribution of RMS injected intravenously into dogs in different body positions, followed by rapid freezing of the postmortem animals. Subsequent transverse band-saw cuts of the frozen thorax were mapped, and selected cores were counted. Following intravenous microsphere injection, Greenleaf et al. (28) inflated postmortem dog lungs to TLC and air-dried the specimen before cutting it into 1-cm transverse slices for gamma camera imaging of each slice. Beck and Rehder (8) also studied microsphere-injected TLC-dried lungs, but they systematically sampled and mapped small cores from transverse slices of the dried specimen. Nicolaysen et al. (45) used circumferentially cut sections from partially inflated and frozen RMS-labeled lungs to refute the existence of a significant radial gradient of flow in the lung. After intravenous injection of a flow label, Glenny and Robertson (25) employed in situ supine tissue fixation and acquired gamma camera images of transverse slices of the fixed lung. Regardless of the approach to fixation, sectioning, and counting, the regional flow heterogeneity attained by these studies revealed coefficients of variation ranging between 20 and 45%. These relatively large flow heterogeneity measurements in normal animals were regarded with some suspicion, because the lung was traditionally considered to have only modest large-scale anatomic gradients of flow attributable to gravity, based on studies utilizing in vivo radionuclide images (56). In retrospect, the 0.3- to 2.5-cm3 resolution attained with the above-cited RMS studies were better than could be attained with in vivo radionuclide images from an earlier era, but the importance of resolution to characterize physiologically relevant regional heterogeneity was not initially appreciated.
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SCALE-DEPENDENT HETEROGENEITY OF LUNG BLOOD FLOW
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As additional high-resolution studies accumulated over time (8, 45), it became clear that some factor unrelated to the gravitational gradient was responsible for a major component of the spatial heterogeneity of regional blood flow. Inspired by a demonstration of scale-dependent heterogeneity of cardiac blood flow (5, 37), a high-resolution study of lung perfusion distribution confirmed that the spatial heterogeneity of regional blood flow increased in a similar predictable fashion as resolution was enhanced (25). This scale dependence of measured regional blood flow heterogeneity can be understood as a consequence of the progressive branching of the pulmonary arterial tree, where flows will be heterogeneous but locally correlated. The relationship between spatial resolution and the spatial heterogeneity of pulmonary blood flow can be characterized by a fractal dimension (27). In a subsequent study using an imaging cryomicrotome to obtain the spatial coordinates of over 75,000 15-µm FMS injected into a rat lung, Glenny et al. (20) were able to characterize regional flow heterogeneity down to an acinar level of scale. Figure 1 from that study illustrates how the logarithm of the coefficient of variation of regional flow increases in a predictable fashion as the logarithm of sampled volume of lung becomes progressively smaller. The slope of the relationship describes how well the flow in one region is correlated with the flow of its neighboring region (27). The steeper curve in Figure 1 demonstrates how this relationship would differ if the distribution of microspheres in the lung were random rather than locally correlated. In agreement with descriptions of a fractal dimension of pulmonary blood flow in several different species (2, 12, 14, 46, 52), this study demonstrated that the extent of measured flow heterogeneity continued to increase as resolution improved, again following a fractal dimension that was not different from the fractal dimension calculated from larger lung volume samples from the same data set. This analysis utilizing microsphere perfusion maps established an important concept relevant to any study of whole organ perfusion: the property of scale-dependent heterogeneity of flow.
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Fig. 1. Plot of the coefficient of variation of regional blood flow in a rat lung as a function of the unit size of lung volume used for the calculation. The slope of the line on this log-log plot describes a spatial fractal dimension (Ds = 1.11) that represents the extent of local correlation of the flow measurements. The steeper slope represents the same calculation made after the regional flow to each volume unit was randomly shuffled, so the flow to one volume unit bears no relation to the flow of its immediate spatial neighbors. By this approach to analysis, the steeper slope with a fractal dimension of 1.50 represents a random distribution of flow. [From Glenny et al. (20).]
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MICROSPHERE STUDIES OF LARGE-SCALE FLOW GRADIENTS IN THE LUNG
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High-resolution microsphere studies have provided important new information concerning the regional changes in pulmonary blood flow attributable to changes in position. Beck and Rehder (7) were the first investigators to specifically delineate high-flow and low-flow regions of the lung whose perfusion was only minimally altered by changes in body position. Glenny et al. (22) compared regional blood flow in prone and supine positions with TLC-dried lungs, demonstrating only minimal shift in per-alveolus blood flow, in contrast to the complete reversal of flow gradients that would be predicted if gravitational force were the only determinant of regional lung blood flow distribution. Because the lungs of larger animals would be expected to show the largest gravitationally determined differences in blood flow, Hlastala et al. (30) conducted a study of pulmonary perfusion in awake horses. For four of the five standing horses, higher regional blood flows were noted in the dorsal caudal part of the lung, opposite of what would be predicted if gravity were the only determinant of large-scale gradients. A similar predominance of posture-independent distribution of blood flow was documented in anesthetized baboons, an animal that is ordinarily upright and has an anatomic structure most similar to human lungs (19).
Investigating gravitational flow gradients by comparison of flows in prone and supine postures is complicated by the compression of dorsal-caudal lung regions in anesthetized animals in the supine posture (33). The influence of gravity on pulmonary blood flow distribution is best studied in a single position, with different G forces as the independent variable. Riding in the National Aeronautics and Space Administration KC-135 aircraft, Glenny et al. (21) administered intravascular FMS to anesthetized pigs experiencing G forces from parabolic flight pathways ranging between microgravity and 1.8 G. The airborne laboratory provided repeated 22-s periods of microgravity, a sufficient time interval to allow the FMS flow labels to be injected and lodge in the microvasculature (36). Figure 2A illustrates a normal extent of regional flow heterogeneity plotted against height up the lung in a prone animal from that study at 1 G. Figure 2B demonstrates the shift in vertical flow distribution for the same animal during microgravity. (For each run, the piece flows were normalized by the mean piece flow for that gravitational condition. Overall cardiac output changed very little among the gravitational interventions.) The large-scale gravitational gradient, indicated by the slopes of the two lines, changes in the expected direction, but the effect is quite small relative to the heterogeneity of flow noted within any isogravitational slice of lung. The study compared prone and supine pulmonary blood flow distribution in microgravity, 1-G, and 1.8-G conditions, documenting large-scale gravitationally directed gradients of flow that increased between microgravity and 1.8-G, although the mean slope differences for each gravitational condition were small. A more comprehensive statistical model incorporating the fixed flow effects of vascular anatomy, posture, gravitational force, and error attributed 85% of the total variability to structure and 2–3% of the observed variability in flow to the difference between microgravity and 1 G. Steeper and statistically significant large-scale gravitationally directed gradients were demonstrable in animal centrifuge studies where larger G forces could be generated (31). Of interest, however, was that even with centrifuge-induced forces of 3–6 G along the head-to-tail z-axis, the addition of a pressure suit to maintain a more normal chest and abdominal wall configuration at high Gz forces minimized the high Gz effect on the slope of the gravitational gradient within the lung (13).
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Fig. 2. A: weight-normalized per-piece blood flow in prone anesthetized pig at normal gravity (1 G), plotted against distance of lung piece from the ventral surface of the lung. B: weight-normalized blood flow for the same lung pieces in the prone pig during microgravity, plotted against distance from ventral surface of the lung. [Data from Glenny et al. (21).]
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The microsphere maps demonstrating both high spatial blood flow heterogeneity and only modest changes in large-scale flow gradients in the lung in response to different postures or gravitational conditions suggested the hypothesis that the anatomy of the pulmonary vasculature is the primary determinant of blood flow heterogeneity and regional blood flow distribution, with gravitational forces contributing a relatively small influence. This hypothesis received additional support from a modeling study by Burrowes and Tawhai (11) that utilized high-resolution measurements of the complete human pulmonary artery tree, predicting regional flow distribution based only on calculations of regional flow resistance calculated from detailed measurements of the pulmonary artery tree. Predicted regional flow distributions were compared with and without the force of gravity. The conclusion from this human pulmonary regional flow model was that the influence of gravity was detectable but quite small relative to the influence of the vascular anatomy. Both the model study of Burrowes and Tawhai and the microsphere maps describe the effect of gravity on per-alveolus blood flow, and they do not include the in vivo effects of gravitationally mediated parenchymal compression. An important difference between microsphere maps and in vivo flow images is that for supine and upright postures, the in vivo maps show higher flows per unit volume in dependent regions primarily because of parenchymal compression (17). In supine anesthetized animals at functional residual capacity, the density difference between nondependent and dependent parenchyma can differ by a factor of three (33). The important insight provided by the microsphere studies is that the large-scale gravitational and postural changes in in vivo blood flow distribution measurements are primarily determined by shifts in parenchymal density, supplemented by a small gravitationally directed contribution to per-alveolus flow in the dependent lung regions.
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EFFECTS OF CHANGES IN CARDIAC OUTPUT ON REGIONAL BLOOD FLOW
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One of the original regional blood flow findings in pump-perfused lung preparations was that flow in the highest part of the lung would increase disproportionately with increases in cardiac output, hence making overall flow more homogeneous (57). Caruthers and Harris (12) provided evidence for this effect with microsphere injections made into pump-perfused sheep lungs over a range of relatively low flows. In intact animals with higher baseline perfusion, however, the change in spatial flow distribution was not apparent. Parker et al. (46) injected microspheres into dogs exercising at a trotting pace, and they showed no difference in overall blood flow heterogeneity between rest and exercise. Bernard et al. (9) used high aerobic-capacity Thoroughbred horses to study the influence of maximal increases in cardiac output on blood flow heterogeneity. They noted no change in overall flow heterogeneity between standing posture and full gallop. Large-scale gradients in the exercising horses remained unchanged, with the highest blood flow consistently delivered to the dorsal caudal portion of the lung during exercise. Melsom et al. (41) measured blood flow distribution in resting and exercising sheep and demonstrated a modest redistribution of blood flow to the dorsal portion of the lung with exercise. Studies of pharmacological manipulation of cardiac output in anesthetized baboons demonstrated no significant differences in flow heterogeneity or large-scale flow gradients (26). Thus, in intact animals, increasing the cardiac output above normal resting levels has little to no influence on spatial pulmonary blood flow distribution. These findings again support the hypothesis that in intact animals with normal or increased cardiac outputs, the anatomic configuration of the pulmonary vasculature is the primary determinant of regional flow distribution.
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TEMPORAL CHANGES IN PULMONARY BLOOD FLOW DISTRIBUTION
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Repeated measurements of flow distribution with injected microspheres have shown good reproducibility, but a component of variability within pieces develops over time, with the largest proportional changes most likely to occur in the low-flow pieces (24). Measurement error also contributes to between-run differences in flow, but the extent of this artifact can be estimated by simultaneous administration of two different RMS or FMS labels (10), with the residual variability attributable to true temporal shifts. For studies of anesthetized dogs and pigs, the contribution of temporal shifts to overall flow heterogeneity has generally ranged between 7 and 17% of the summed heterogeneity (2, 23, 24). In response to the question of whether any structure could be discerned within these temporal shifts, Glenny et al. (24) applied the statistical technique of cluster analysis to regional pulmonary blood flow measurements in anesthetized dogs. They demonstrated that these small spontaneous temporal shifts in regional flow were not random, but rather they took place among lung pieces adjacent to each other. As might be expected from a branching distribution system, when one region gained in relative flow, its immediate neighbor tended to lose flow. The size of the units undergoing these spontaneous shifts was larger than the 2-cm3 piece size, generally in the 20-cm3 range. Some fraction of the temporal variability observed in anesthetized animals may have been attributable to effects anesthesia or mechanical ventilation, because sequential FMS measurements of regional pulmonary blood flow in awake, standing dogs over 1 wk showed a temporal coefficient of variation of only 7% (23). In this study of awake animals, the lung pieces with the greatest variability still tended to be the low-flow pieces, but the awake animals generally demonstrated less temporal variability than that previously described with anesthetized animals.
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PULMONARY VASCULAR RESPONSE TO HYPOXIA
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While the pulmonary vascular hemodynamic response to hypoxia has been studied for decades, the issue of whether there might be regional differences in response had not been apparent from in vivo imaging studies. Melsom et al. (41) first demonstrated consistent region-dependent changes in blood flow in response to hypoxia in awake sheep. Hlastala et al. (32, 39, 53) investigated the regional blood flow response to different levels of hypoxia in both anesthetized dogs and pigs. Applying the cluster analysis approach previously utilized in the temporal change study cited above, Hlastala et al. were able to demonstrate relatively stereotypical regional changes in flow distribution among all animals, with specified regions receiving more blood flow during hypoxia and other regions receiving less flow. These animal findings were relevant to the interpretation of the human high-altitude pulmonary edema, where a heterogeneous pulmonary vascular response to hypoxemia has been postulated to explain the patchy nature of the pulmonary edema observed in that condition.
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MICROSPHERE AEROSOLS TO MAP REGIONAL VENTILATION
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Zeltner et al. (58) administered a dilute aerosol of 0.9-µm FMS to guinea pigs, and they documented with histology that the microsphere deposition was almost exclusively alveolar. This suggested that an FMS aerosol in that size range might be used to produce maps of regional ventilation, provided an adequate fluorescence signal could be obtained from postmortem cut lung pieces. Both Melsom et al. (43) and Robertson et al. (49) found that a 5-min administration of an inhaled 1.0-µm FMS aerosol to goats or pigs yielded an adequate fluorescent signal from 2-cm3 pieces of air-dried lung. Measurements utilizing simultaneous administration of different FMS aerosol labels demonstrated reproducibility that was comparable to the earlier regional blood flow measurements made with the 15-µm injected FMS. The aerosol deposition on large conducting airways was minimal relative to the total fluorescence signal in the lung, so the proportional deposition of fluorescent label among the subsequently cut pieces appeared to be an appropriate estimate of regional alveolar ventilation (47). These measurements of regional ventilation demonstrated that the heterogeneity of ventilation was comparable to that described for regional blood flow, at least down to the level of the 2-cm3 piece size chosen for these studies. An important consequence of development of the FMS aerosol method for measurement of regional ventilation was that when paired with simultaneous injection of intravenous 15-µm FMS labels, high-resolution maps of ventilation-perfusion (A/) distribution could be created.
The validity of aerosol deposition measurements to represent high-resolution maps of ventilation remains to be firmly established. Although Melsom et al. (43) demonstrated equivalent microsphere ventilation map measurements with the 1.0-µm microspheres and the ultrafine particles of Technegas, even the smallest aerosol will distribute differently from gas molecules at an alveolar level of scale (29). While the highest resolution where aerosol deposition can still be interpreted as a valid measure of regional ventilation remains to be determined, the results from FMS ventilation maps at the 2-cm3 level of scale can be compared with other high-resolution methods for regional ventilation measurement. Kreck et al. (38) developed a regional ventilation model that utilized sequential computed tomography (CT) lung images acquired during the washin of 65% xenon to create a high-resolution a map of regional ventilation. Robertson et al. (50) compared regional FMS ventilation measurements in five anesthetized sheep with measurements with the CT xenon model described by Kreck et al. (38), where the CT analysis volume was adjusted to the 2-cm3 size of the cut lung pieces. Because the CT images were acquired with sheep imaged in the supine posture, there were minor lung shape discrepancies, but the overall extent of heterogeneity described by the two methods was comparable, and large-scale gradients of ventilation were likewise comparable. Hence at least at the 2-cm3 level of scale, the aerosol microsphere maps of regional ventilation agree reasonably well with an independent technique, but confirmation of these findings at a higher level of resolution is still needed.
The ventilation maps obtained using the FMS aerosol allowed exploration of the small-scale and large-scale properties of regional ventilation. Altemeier et al. (1) performed fractal analysis of ventilation maps of prone pigs and demonstrated a consistent scale-dependent progression in spatial heterogeneity as the volume analyzed became smaller. The fractal dimension calculated from this ventilation data was comparable to the fractal dimension calculated for regional blood flow, confirming that the rate of increase in spatial heterogeneity with decreasing scale was comparable for the two parameters. One appreciable difference between microsphere ventilation and perfusion maps has been noted in large-scale differences in regional ventilation between prone and supine postures. Altemeier et al. (2) showed that there was a large-scale decrement in dorsal-caudal aerosol deposition in the anesthetized supine pig that was not noted in the prone position. This marked regional decrease in ventilation was associated with a lesser dorsal-caudal decrement in perfusion that accounted for the increased hypoxemia noted in the supine posture. This study described the disproportionate gas-exchange influence exerted by the supine compression of the dorsal-caudal portion of the lung in anesthetized animals, an issue that had not been separately analyzed in earlier prone-supine comparisons of regional blood flow (22, 40).
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FMS STUDIES OF A/ DISTRIBUTION
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Simultaneous FMS measurements of both regional ventilation and regional blood flow have clarified how the high extent of heterogeneity noted in both parameters could still yield normal gas exchange. For animals examined at a fixed level of scale, there is appreciable variability among animals in the extent of spatial heterogeneity of regional ventilation and regional perfusion, but for any single animal, the extent of heterogeneity of the two parameters is roughly comparable. Within animals, the correlation between regional ventilation and regional blood flow has ranged between 0.76 and 0.92 (1, 49) with the higher correlations noted in those animals with the greatest overall extent of alveolar ventilation and blood flow heterogeneity. Figure 3 illustrates the range of heterogeneity of both ventilation and blood flow and the tight correlation between the two parameters in a prone anesthetized pig from the study of Robertson et al. (49). Melsom et al. (43) demonstrated comparable heterogeneity and correlations between alveolar ventilation and blood flow in microsphere measurements in awake goats.
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Fig. 3. Correlation between regional blood flow and regional ventilation in a prone anesthetized pig. [From Robertson et al. (49).]
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Because a A/ measurement is made for every piece of the cut lung, calculations utilizing the A/ distribution, measurements of mixed venous blood gases, cardiac output, and ventilation can be used to predict gas exchange. Altemeier et al. (3) studied normal anesthetized pigs, making FMS measurements of alveolar ventilation and blood flow and also acquiring gas- exchange data using the multiple inert-gas elimination technique (MIGET) (55). The A/ distributions derived from FMS and MIGET techniques showed excellent agreement, with both calculated distributions able to predict arterial blood- gas values. Furthermore, utilizing the FMS A/ distributions and the mixed venous inert gas concentrations, Altemeier et al. (3) were able to accurately predict the individual inert-gas retention and excretion values as an additional validation of the concurrence between the FMS A/ distributions and gas- exchange characteristics of the lungs. However, the agreement between calculations based on FMS A/ distributions and MIGET measurements was weaker in both a bead embolization injury model (4) and an endotoxin injury model (16). For both of those studies, the calculations based on FMS data underestimated the extent of gas-exchange abnormality, and the authors postulated that the experimental injuries produced a spatially distributed injury that could not be adequately represented by the FMS method because injury was heterogeneous within the 2-cm3 lung piece size.
The comparison between FMS A/ distributions and MIGET A/ distributions in normal animals (3) used FMS data with 2-cm3 pieces of lung. While the heterogeneity of perfusion is scale dependent below the resolution of that study, the agreement of results between techniques for normal lungs suggests that overall A/ heterogeneity may not exhibit the same scale-dependent property shown by its two components. It seems plausible that the heterogeneity of ventilation at smaller scales may increase in parallel with the heterogeneity of perfusion but that the correlation between the two parameters remains stable and scale independent. While ventilation heterogeneity has not been measured at a scale smaller than 2 cm3, Robertson et al. (48) applied the fractal ventilation data set of Altemeier et al. (1) to examine a range of different volumes of regional ventilation and perfusion measurements in the same animal. Although the heterogeneity of both alveolar ventilation and blood flow increased as the lung was subdivided into smaller and smaller volumes, the overall A/ distribution did not broaden. Hence at least analysis of data down to the 2-cm3 level of scale shows that A/ heterogeneity does not demonstrate the scale-dependent properties of its two components or that the correlation between alveolar ventilation and blood flow in a normal lung remains relatively constant regardless of the resolution attained for the maps of flow.
Studies of the posture-related large-scale shifts in regional A/ using microsphere techniques should yield similar findings to in vivo A/ imaging methods because in vivo tissue compression will affect regional measurements of both alveolar ventilation and blood flow equally. Mure et al. (44) utilized combined FMS measurements of ventilation and blood flow in prone and supine positions to show that A/ distribution was more uniform in the prone position. Utilizing multiple measurements performed in prone and supine positions in anesthetized pigs, Altemeier et al. (2) showed that regional per-alveolus ventilation was appreciably reduced in the dorsal-caudal regions of lung in the supine position. That study also established the respective quantitative contributions of posture, time, and experimental error to the heterogeneity of both regional ventilation and regional blood flow. As with earlier studies, the major contribution to regional heterogeneity of both alveolar ventilation and blood flow was fixed or anatomic, comprising 74 and 63% of the total heterogeneity, respectively, with posture contributing 16 and 24% and temporal changes accounting for the balance.
Given the heterogeneity of regional alveolar ventilation and blood flow in a normal lung, a simple mechanism to explain the strong match of the two parameters would be to propose that the heterogeneity of ventilation is a fixed property established during growth and that active pulmonary vasoconstriction adjusts flow to those fixed characteristics. This hypothesis was tested by Melsom et al. (42), utilizing microsphere maps to characterize A/ distributions in awake sheep before and after inhalation of nitric oxide. They noted no significant change in A/ distribution after nitric oxide inhalation. Glenny et al. (26) studied the distribution of perfusion alone in normal anesthetized baboons before and after the infusion of PGI2, a potent pulmonary vasodilator, and showed no appreciable change in perfusion distribution or alveolar-arterial O2 difference. While these findings appear to exclude simple vasoconstriction as the mechanism tuning blood flow to alveolar ventilation in a normal lung, it still remains possible that long-sustained pulmonary vasoaction might trigger morphological adjustments in vascular structure to account for the normal degree of A/ homogeneity.
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DIRECTIONS FOR FUTURE INVESTIGATION
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The high-resolution microsphere flow maps of normal lung have revealed an unexpected extent of ventilation and blood flow heterogeneity in an organ that appears relatively uniform at a microscopic level. This heterogeneity is spatially organized so that ventilation and blood flow remain tightly matched, as required for normal gas exchange. High-resolution in vivo images of the pulmonary arterial tree are being incorporated in flow models to suggest that the anatomy of the pulmonary arterial tree may be the primary determinant of this blood flow heterogeneity. However, these anatomically based modeling studies have yet to be linked to data acquired with microsphere flow maps, and this step will be essential to strengthen the hypothesis that regional blood flow heterogeneity has a primary anatomic explanation.
Explaining the basis of ventilation heterogeneity is more complex than perfusion heterogeneity because regional ventilation is influenced both by conducting airway anatomy and local compliance. Studies with pleural capsules (15) and implanted lung markers (35) have demonstrated substantial small-scale variability in local compliance. Although it seems likely that local airways conductance and local compliance are correlated, this has not been directly demonstrated. Hence, model studies of regional ventilation based on airway dimensions alone are not likely to agree well with microsphere maps, but those maps together with airway model predictions may provide some insight into the correlation between local compliance and airways conductance. Finally, the aerosol ventilation maps are of uncertain validity at higher resolution, for at some yet-to-be-determined level of scale, the extent of 1.0-µm aerosol deposition will fail to represent local ventilation. Better estimates may come from smaller aerosols, and FMS particles in the 0.04-µm range, now commercially available, may provide additional insight into high-resolution regional ventilation.
Microsphere A/ maps have been applied to predict gas exchange abnormalities in pulmonary embolus and pulmonary edema models, but calculations based on the maps have underestimated the extent of hypoxemia (4, 16, 54). It appears that higher resolution maps will be required to appropriately predict the gas-exchange consequences of diffuse lung injury. In addition, most current studies of disease models utilize small animals, again requiring high-resolution data. The cryomicrotome system used to produce the acinar-level maps of regional blood flow in rat lungs (20) can image deposited FMS aerosols, offering the potential for high-resolution ventilation measurements. However, the aerosol particles are far below the resolving power of the cryomicrotome system, and each cryomicrotome aerosol image is homogenized by signal from aerosol deeper within the specimen. To date, no correction for this homogenizing effect has been developed to provide accurate local measurements of aerosol deposition. Hence new approaches are needed for analysis of microscopic aerosol images to provide valid high-resolution A/ maps for small animal disease models and for studies of mechanisms of A/ matching during growth.
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GRANTS
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Preparation of this review was supported by National Heart, Lung, and Blood Institute Grants HL-24163 and HL-073598.
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FOOTNOTES
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Address for reprint requests and other correspondence: H. T. Robertson, Pulmonary and Critical Care Medicine, Box 356522, Univ. Hospital, Seattle, WA 98195-6522 (e-mail: tomrobt{at}u.washington.edu)
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REFERENCES
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ADVANTAGES OF MICROSPHERE MAPS
