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J Appl Physiol 82: 678-685, 1997;
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Journal of Applied Physiology
Vol. 82, No. 2, pp. 678-685, February 1997
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Pulmonary blood flow distribution has a hilar-to-peripheral gradient in awake, prone sheep

Sten M. Walther2, Karen B. Domino1, Robb W. Glenny3, Nayak L. Polissar3, and Michael P. Hlastala2,3

Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Medicine, University of Washington School of Medicine, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Walther, Sten M., Karen B. Domino, Robb W. Glenny, Nayak L. Polissar, and Michael P. Hlastala. Pulmonary blood flow distribution has a hilar-to-peripheral gradient in awake, prone sheep. J. Appl. Physiol. 82(2): 678-685, 1997.---We examined the pulmonary blood flow distribution with intravenous fluorescent microspheres (15 µm) in nine prone, unanesthetized, lambs. Lungs flushed free of blood were air-dried at total lung capacity and sectioned into ~2-cm3 pieces. The pieces were weighed, identified by lobe, and assigned spatial coordinates. Fluorescence was read on a spectrophotometer, and signals were corrected for piece weight and normalized to mean flow. Pulmonary blood flow heterogeneity was assessed by using the coefficient of variation of the flow data. The number of pieces (±SD) analyzed were 1,249 ± 150/animal. Heterogeneity of blood flow was 29.5 ± 6.5% (coefficient of variation = SD/mean). Pulmonary blood flow decreased with distance from hilus (P < 0.002) but did not change significantly with vertical height. Distance from the hilus was the best predictor of pulmonary blood flow (R2 = 0.201) and, together with spatial coordinates and lobe, accounted for 33.7 ± 12.0% of blood flow variability. We conclude that pulmonary blood flow in the awake, prone sheep is distributed with a hilar-to-peripheral gradient but no significant vertical gradient.

lung; regional pulmonary blood flow; heterogeneity; gravitational gradient; methods; fluorescence; microspheres;

INTRODUCTION

GRAVITY HAS TRADITIONALLY been thought to be the major determinant of the pulmonary blood flow distribution. This concept was based on studies showing increased blood flow in dependent lung regions in humans and animals by using two-dimensional imaging of radiolabeled gases in the lung (1, 2, 19, 21, 34). More sensitive techniques utilizing images of embolized microspheres or macroaggregates of proteins (24, 26, 27) and imaging with positron-emission tomography (5, 29) also showed such topographic inequalities in pulmonary blood flow distribution.

The importance of gravitational factors in determination of the distribution of pulmonary blood flow has recently been challenged. Gravity-independent ventral-to-dorsal and cranial-to-caudal flow gradients have been reported in anesthetized dogs (28), horses (8, 18), and ponies (20), and in isolated dog lungs (4). Glenny et al. (11), analyzing the three-dimensional distribution of microspheres injected in different postures in halothane-anesthetized dogs, found increased flow in dorsal regions independent of posture. Gravity accounted for only 5.8 and 2.4% of flow variability in the supine and prone positions, respectively. Using positron-emission tomography, Treppo et al. (33) also did not observe a systematic gravitational gradient in anesthetized and mechanically ventilated, prone dogs.

In addition, gravity-independent central-to-peripheral flow gradients have been observed in humans (17), dogs (11, 14-16, 27), and ponies (20) by using both high-resolution imaging and microsphere techniques. The increased pulmonary blood flow in central lung regions suggests that vascular anatomic factors are important in determination of the blood flow distribution. A radial gradient is consistent with the fractal branching pattern of the pulmonary circulation (12), where vascular resistance is increased by branching and longer circuit pathways (4, 12, 15-17, 27).

Most studies that provide detailed images of perfusion distribution have been performed in dogs, a species with extensive pathways for collateral ventilation (35). The architecture of pulmonary arteries differs among species with different collateral ventilation. Animals with low resistance to collateral airflow rely less on hypoxic pulmonary vasoconstriction for ventilation-perfusion matching, and, consequently, have thin-walled pulmonary arteries (22). Conversely, animals with little or no collateral ventilation, such as pigs (35), have a vigorous vasoconstrictive response to alveolar hypoxia (22). We hypothesized that differences in pulmonary vasoreactivity could influence the distribution of perfusion in the lungs. Pigs may have a pulmonary blood flow distribution less dependent on anatomic influences than that of dogs. Because pigs are difficult to handle and study in an unanesthetized state, the present study was undertaken in the awake, spontaneously ventilating lambs, which have been shown to have little collateral ventilation (32). We found less blood flow heterogeneity than previously described in dogs, and a hilar-to-peripheral, but not a vertical dorsal-to-ventral, gradient.

METHODS

Animal preparation. The study was approved by the University of Washington Animal Care Committee. Nine healthy lambs [four females and five males; weight = 20.8 ± 1.5 (SD) kg] were used. The lambs were fasted overnight but had free access to water. Without premedication, the animals were suspended in a sling in the prone position with their legs protruding through holes, allowing free movement of the neck and head. The sling was shallow to allow for unrestrained motion of the thorax. With the animals in this position, an intravenous catheter was introduced into a peripheral foreleg vein.

Pulmonary blood flow determination. Pulmonary blood flow was determined as described by Glenny et al. (10). One of six colors (red, orange, yellow-green, crimson, blue, and blue-green) of 15-µm fluorescent latex microspheres (FluoSpheres, 0.2% solids, Molecular Probes, Eugene, OR) was randomly used in each experiment. The microsphere suspension was sonicated for 5 min and vortexed immediately before slow injection (>60 s) of 2 ml (3 ml for crimson) through the intravenous cannula (2-3 × 106 microspheres). The catheter was thoroughly flushed with saline after the injection.

The animals were anesthetized with pentobarbital sodium, and the trachea was intubated and connected to a piston-ventilator (Harvard, South Natick, MA). Two (right and left) femoral polyethylene arterial cannulas and a triple-lumen femoral vein cannula were introduced through cut downs. A rapid intravenous infusion of saline was started. The animals were heparinized (20,000 U) and exsanguinated through the arterial cannulas. After withdrawal of ~50% of the blood volume, papaverine (60 mg iv) was given to vasodilate the pulmonary vasculature and facilitate flushing of the lungs. A median sternotomy was performed, and the pulmonary artery and left atrium were cannulated with wide-bore catheters. A 2% dextran solution was infused by means of gravity (15-20 cmH2O) into the pulmonary circulation until the effluent from the left atrium was clear of blood. The lungs were excised, the trachea was connected to a pressure source, and the lungs were reinflated to total lung capacity (constant airway pressure of ~25 cmH2O) and suspended to dry. The lungs were punctured 20 times with a 20-gauge 15-cm needle to improve drying. A pilot study demonstrated incomplete drying and decomposition of central lobar lung tissue in sheep lungs when needle puncture was not performed. The anatomic configuration of the lungs was preserved by gluing together the apical and the most ventral rims of the left and right lungs with a small amount of cyanoacrylate glue (Duro Superglue, Loctite, Cleveland, OH).

After drying for 6-8 days, the lungs were coated with a 1-cm-thick layer of polyurethan foam (Kwik Foam, DAP, Dayton, OH). The foam-enveloped lungs were suspended in a plastic-lined square box so that their intrathoracic position was reproduced. The box was filled with rapidly setting urethan foam (Polyol and isocyanate, International Sales, Seattle, WA). The foam block was sliced into 1.2-cm-thick slices with a band saw with a blade designed to eliminate tearing and loss of tissue, and the slices were cut into squares (1.2 × 1.2 cm) to yield cubes ~1.7 cm3 in volume. Foam adhering to lung tissue was removed, and the pieces were weighed. Samples with a weight <0.008 g were discarded, and the remaining pieces were identified by lobe and assigned unique x, y, and z coordinates, where x represents distance in sagittal planes (left-right), y represents distance in coronal planes (dorsal-ventral), and z represents distance in transverse planes (caudal-to-cranial), respectively. The number of airways present in the pieces was estimated (<10% of piece volume; 10-25%, 25-50%, 50-75%, >75%, trachea or part of trachea) and coded.

Fluorescent dye was extracted from lung tissue samples by soaking in 1.50 ml of 2-ethoxyethyl acetate (Cellosolve, Aldrich Chemical, Milwaukee, WI) for 48 h. The supernatant was pipetted into cuvettes and read in a fluorescent spectrophotometer (model LS 50B, Perkin Elmer, Norwalk, CT) at the dye-specific excitation emission wavelengths.

A kidney was harvested from each animal, and a 1- to 2-g section of the cortex was digested for 24-48 h in 4N KOH, after which it was filtered through a 10-µm-pore polycarbonate filter (Poretics, Livermore, CA). The filter containing the microspheres was soaked in Cellosolve for 4 h, and the fluorescence from the supernatant was measured.

Statistical methods. Tissue samples with an airway content of 25% or more were not included in the final analysis. Fluorescence was corrected for the weight of each piece by dividing by weight. Volume-normalized relative pulmonary blood flow to each piece of lung was calculated by dividing the fluorescence to each piece by the mean fluorescence of all pieces. Right, left, and lobar normalized blood flows were summed and expressed as percentages of total flow. The pulmonary hila were defined with spatial coordinates as the points of entry of the left and right pulmonary arteries into the lungs (9). The radial distance in centimeters from the ipsilateral hilus (dh) for a piece with coordinates (x, y, z) was calculated by the usual Euclidean distance formula
d<SUB><IT>h</IT></SUB> = 1.2 &z.ccirf; <RAD><RCD>(<IT>x</IT> − <IT>x</IT><SUB><IT>h</IT></SUB>)<SUP>2</SUP> + ( <IT>y</IT> − <IT>y</IT><SUB><IT>h</IT></SUB>)<SUP>2</SUP> + (<IT>z</IT> − <IT>z</IT><SUB><IT>h</IT></SUB>)<SUP>2</SUP></RCD></RAD>
where the subscript h indicates coordinates for the hilus, and the factor 1.2 was used to convert the dimensionless distance to centimeters.1

The coefficient of variation (SD/mean) was used to describe the heterogeneity of flow in the whole lung, within the right or left lung, and within planes and lobes. Differences in heterogeneity between sagittal, coronal, and transverse planes and between lobes were analyzed with repeated measures analysis of variance. When variation across these categories was statistically significant, post hoc comparisons were made by using the method of least significant difference.

Normalized flow was characterized as a linear function of x, y, and z spatial coordinates or distance from the ipsilateral hilus by using least squares regression analysis. A slope of -4.0%/cm, for example, means that flow decreases 0.04 normalized flow units/cm. The slope can be expressed in more familiar terms as a percentage per centimeter because the mean normalized flow for the entire animal is 100%. The mean slopes (e.g., flow vs. x) were compared with zero with a single-sample two-tailed t-test. Pearson's correlation (r) was used to quantify the strength of the relationship between flow and other variables in each animal, and R2 was used to express the proportion of pulmonary blood flow variability that was explained by the independent variables. A single-sample two-tailed t-test using Fisher's Z transformation for r (30) was performed to test whether correlations were significantly different from zero.

To assess the hilar-to-peripheral and gravitational gradients within a transverse section, new slopes of blood flow vs. x, y, and distance from the hilus were recalculated after correction for trends in the caudal-cranial (z) direction. This analysis describes how much of these gradients are in the caudal-cranial direction. Because blood flow in direction z tended to be curvilinear rather than linear (due to increased flow in the hilar regions), we fitted a quadratic polynomial to flow as a function of z. The new slopes, after adjustment for z, were calculated in simple linear regression models for residual flow as a function of either x, y, or radial distance. Residual flow was calculated as observed minus fitted flow values from the fitted quadratic model based on z. These slopes were also compared with zero with a single-sample two-tailed t-test.

RESULTS

The number of pieces analyzed and their weights and number of planes per animal are summarized in Table 1. Kidney samples did not have any fluorescence, indicating that all of the microspheres were trapped in the lungs.

Table 1. Number of pieces, piece weight, and planes per animal

Sheep No. Weight, kg Total Number of Pieces No. of Pieces Excluded* No. of Pieces Analyzed Piece Weight, mg Sagittal Planes Transverse Planes Coronal Planes
1 20.0 1,374 64 1,310 35 ± 14  16 23 14
2 20.0 1,138 87 1,051 35 ± 18  14 17 13
3 23.5 1,347 102 1,245 32 ± 14  16 20 14
4 22.5 1,126 70 1,056 37 ± 18  15 21 14
5 19.0 942 71 871 38 ± 21  15 18 11
6 19.0 1,225 60 1,165 36 ± 16  15 21 13
7 21.0 1,282 61 1,221 34 ± 22  14 21 13
8 22.0 1,454 111 1,343 29 ± 12  17 21 15
9 20.0 1,356 132 1,224 32 ± 13  16 22 15
Mean ± SD 20.8 ± 1.5  1,249 ± 150  84 ± 25  1,165 ± 140  34 ± 3  15 ± 1  20 ± 2  14 ± 1
* More than 25% airways (see METHODS).

Total relative flow per side and lobe is shown in Table 2. Pulmonary blood flow was always larger to the right lung, and the right-to-left lung blood flow ratio was typically 3:2. Mean heterogeneity of pulmonary blood flow was 29.5 ± 6.5 (SD)% (Table 2). There were generally a substantial number of low-flow pieces, resulting in an asymmetric distribution with a mean skewness of -0.18 ± 0.65 (see example of perfusion distribution, Fig. 1). Flow heterogeneity was similar on both sides and in all lobes with the exception of the right apical lobe, which had a significantly larger heterogeneity than the other lobes (P < 0.01, Table 2). The large flow heterogeneity in the right apical lobe was due to contiguous regions with low normalized flows in the ventral and most cranial parts of the lobe in five animals. Calculation of pulmonary blood flow heterogeneity within each plane and that averaged across planes revealed that pulmonary blood flow heterogeneity was smallest for sagittal planes, although not significantly different from the coronal or transverse planes.

Table 2. Relative pulmonary blood flow and pulmonary blood flow heterogeneity as expressed by coefficient of variation per side and lobe

Total Flow, %  Heterogeneity, % 
Left lung 42.5 ± 3.6  27.0 ± 8.5 
  Apical lobe 13.5 ± 2.1  23.9 ± 6.1 
  Diaphragmatic lobe 29.1 ± 4.9  26.6 ± 8.6 
Right lung 57.5 ± 3.6  30.7 ± 5.9 
  Apical lobe 12.1 ± 4.3  40.9 ± 16.8*
  Diaphragmatic lobe 31.1 ± 4.0  25.9 ± 8.5 
  Accessory lobe 4.9 ± 3.7  25.0 ± 8.2 
  Middle lobe 9.4 ± 3.5  26.9 ± 6.6 
Whole lung 100 29.5 ± 6.5
Values are means ± SD; n = 9 sheep. Total flow data were not tested statistically. * Significantly different from other lobes, P < 0.01 (repeated-measures ANOVA, post hoc test based on least significant difference).

Fig. 1. Frequency distribution of normalized pulmonary blood flow in 1 lamb. Total no. of pieces = 1,310, SD = 0.295, skewness = -0.83. Flow intervals are 5% of mean flow.
[View Larger Version of this Image (29K GIF file)]

Blood flow decreased linearly with increasing distance from the ipsilateral hilus (Table 3 and an example in Fig. 2). Pulmonary blood flow decreased 4.0 ± 2.5%/cm as a linear function of distance from the ipsilateral hilus [mean (±SD) slope = 4.0 ± 2.5%/cm, 95% confidence interval = (-5.9 and -2.2%/cm), P < 0.002 compared with zero]. The hilar-to-peripheral blood flow ratio, calculated from the hilar-to-peripheral slope for each animal multiplied by the distance from the hilus to the periphery (which varied from ~8 to 16 cm), was 1.5-2.8:1.

Table 3. Pulmonary blood flow as a linear function of distance from ipsilateral hilus, vertical height, caudal-cranial height, and left-right vectors

Sheep No. Slopes
Distance from hilus, %/cm Dorsal-ventral vector, %/cm Caudal-cranial vector, %/cm Left-right vector, %/cm
1  -4.1  -0.9  -0.5 0.9
2  -2.1  -1.2 0.9 1.0
3  -6.8 2.5 3.8 1.3
4  -0.3  -0.6  -1.0  -0.5
5  -2.6  -0.6 0.7  -0.8
6  -5.8 1.3 2.7  -0.6
7  -3.3  -1.2 0.5 0.4
8  -3.3 0.3 1.9 1.3
9  -8.1 4.7 4.1 0.9
Mean ± SD  -4.0 ± 2.5*  -0.5 ± 2.0  1.5 ± 1.8dagger 0.4 ± 0.8 
95% CI (-5.9, -2.2) (-1.1, 2.0) (0.1, 2.8) (-0.2, 1.1)
CI, confidence interval. * Significantly different from zero, P = 0.002 (t-test). dagger Significantly different from zero, P = 0.04 (t-test).

Fig. 2. Normalized pulmonary blood flow as a function of distance from ipsilateral hilus of a representative lamb. Hilar-to-peripheral gradient is shown, with a decrease in flow of 4.1%/cm. Least squares linear regression: pulmonary blood flow = 1.3-0.041 × distance from ipsilateral hilus, R2 = 0.17. 
[View Larger Version of this Image (37K GIF file)]

Distance from the ipsilateral hilus was the strongest single predictor of pulmonary blood flow, accounting for 20.1 ± 15.0% of the flow variance in the whole lung. When analyzed by lobe, the variance explained by hilar distance was less in the left apical lobe (mean R2 = 5.3%) and higher in the left and right diaphragmatic and right apical lobes (mean R2 = 30.0, 26.7, and 25.8%, respectively). The slope of flow vs. hilar distance was also examined after exclusion of lung pieces with a weight <1 SD below mean weight in each animal, with a similar result (mean slope = 3.8%/cm). The hilar-to-peripheral gradient persisted within transverse planes, after correction for trends in the caudal-cranial (z) direction. However, it was smaller than the uncorrected slope (P < 0.004, compared with uncorrected slope), with a mean slope that was -0.7 ± 0.8%/cm (P = 0.04 compared with zero).

In contrast, overall pulmonary blood flow was not affected by the gravitational (dorsal-to-ventral) gradient (Table 3 and an example in Fig. 3A). The mean slope of pulmonary blood flow as a linear function of vertical height was not significantly different from zero [-0.5 ± 2.0%/cm, 95% confidence interval = -1.1 and 2.0%/cm)]. This slope remained unchanged and not significantly different from zero after correction for trends in the cranial-caudal direction (-0.7 ± 1.4%/cm). When analyzed separately by lobe, the right and left diaphragmatic lobes exhibited a small but increasing flow along the gravitational vector (mean R2 = 0.064 and 0.104, respectively). However, regression analysis revealed that this could be explained by a larger blood flow dependence on the caudal-cranial vector and not an independent effect of gravity.

Fig. 3. Normalized pulmonary blood flow distribution for coronal (A), transverse (B), and sagittal (C) planes in a representative lamb. Note lack of gravitational (dorsal-to-ventral) gradient (A) and presence of a central-to-peripheral gradient (B). The 45 of 50 pieces with a flow <0.25 were located in most cranial and ventral portions of right apical lobe.
[View Larger Version of this Image (18K GIF file)]

Caudal-to-cranial gradients had a curvilinear appearance, with the mode usually close to the level of the hilus (Fig. 3B). Blood flow increased linearly with increasing distance along this vector (Table 3, mean (±SD) slope = 1.5 ± 1.8%/cm, P = 0.04, compared with zero). The quadratic equation for this flow had mean constant, linear, and quadratic coefficients of 0.394, 0.118, and -0.0049, respectively. Left-right gradients in pulmonary blood flow generally had a bimodal shape, with lower flow numbers in the most lateral and medial sagittal planes (Fig. 3C). The slope (0.4 ± 0.8%/cm) was not significantly different from zero (Table 3). This slope changed very little after correction for trends in the cranial-caudal direction (0.5 ± 1.0%/cm).

Among correlations (Table 4) between pulmonary blood flow and x, y, and z spatial coordinates and distance to the ipsilateral hilus, there was a significant negative correlation (P = 0.002) between blood flow and distance to ipsilateral hilus (r = -0.41 ± 0.20, Table 4). There was also a significant positive correlation between blood flow and the caudal-cranial vector (r = 0.25 ± 0.28, P = 0.03) because of an increased flow in the middle areas at the level of the hilum. In contrast, there was little association between left-right and dorsal-ventral vectors and pulmonary blood flow (Table 4).

Table 4. Linear association (Pearson's correlation coefficients) between pulmonary blood flow and spatial vectors for individual animals

Sheep No. Distance From Hilus DorsalVentral Vector CaudalCranial Vector Left-Right Vector
1  -0.42  -0.10  -0.10 0.13
2  -0.25  -0.14 0.16 0.15
3  -0.62 0.24 0.55 0.15
4  -0.04  -0.07  -0.15  -0.07
5  -0.31  -0.08 0.15  -0.13
6  -0.65 0.16 0.54  -0.09
7  -0.32  -0.13 0.08 0.05
8  -0.50 0.05 0.44 0.23
9  -0.60 0.37 0.54 0.08
Mean ± SD  -0.41 ± 0.20* 0.03 ± 0.18  0.25 ± 0.28dagger 0.06 ± 0.13 
95% CI (based on Fisher's transformation) (-0.64,     -0.27) (-0.11,   0.18) (0.03,     0.51) (-0.04,   0.16)
* Significantly different from zero, P = 0.002 [t-test of Fisher's Z transformation for Pearson's correlation coefficient (r)]. dagger Significantly different from zero, P = 0.03 (t-test of Fisher's Z transformation for r).

Multiple regression, including regional flows from all animals, revealed that the largest part of flow variance was contributed by the hilar-peripheral vector. The left-right vector and identification by lobe added minimally to the explanation of flow variance. Other vectors (caudal-cranial and dorsal-ventral) contributed very little. The contribution to the flow variance of anatomy, as defined by lobe and linear functions of x, y, and z spatial coordinates, was 26.5 ± 14.2% [(mean ±SD) R2, range = 6.6 to 47.2%]. When distance to ipsilateral hilus was added to the multiple regression, the explained flow variance increased to 33.7 ± 12.0%. This distance alone explained 20.1 ± 15.0% of flow variance.

DISCUSSION

The major finding of this study was that pulmonary blood flow in normal, unanesthetized, spontaneously breathing sheep was distributed with a hilar-to-peripheral gradient but with no significant vertical gradient in the prone position.

Methodological issues. To estimate regional pulmonary blood flow reliably, the fluorescent microsphere method has to fulfill a number of criteria. First, the spheres have to be totally extracted by the pulmonary microcirculation. This was accomplished in the present experiment as indicated by the absence of fluorescence in the kidney samples. Second, to faithfully represent regional blood flow in the lungs, the microspheres must have a distribution in the microcirculation similar to that in whole blood. This issue was recently evaluated by using 15-µm radiolabeled microspheres and a radiolabeled "molecular microsphere," hydroxy-iodobenzyl-propanediamine (25), which is almost completely extracted from the plasma phase during the first passage through the lungs. Distribution of the microspheres and hydroxy-iodobenzyl-propanediamine were highly correlated in small regions (~1.5 cm3) in the goat lung. Although these authors used radiolabeled microspheres, 15-µm fluorescent microspheres were recently validated for measurement of regional pulmonary blood flow (10). Third, the number of microspheres have to be large enough to limit the effect of method error on the statistics being calculated. We injected 2-3 × 106 microspheres, which were calculated to assure a sufficient number (roughly 400) per tissue sample when blood flow was 25% of average flow (6).

Because our focus was to examine flow gradients and the relative flow distribution, flows in each lung piece were normalized to the mean flow of all pieces per animal. Flow signals were corrected for piece weight because a substantial number of tissue pieces from peripheral parts of the lungs had a volume of <1.7 cm3. However, pieces with airways and vessels are relatively heavy compared with the lung parenchyma. As they contribute substantially to the weight of the sample, inclusion of airways would result in erroneously low weight-corrected pulmonary blood flow. To minimize this artifact, major vessels and airways were visually dissected from the lung parenchyma before processing. In addition, lung pieces with >25% airways (7% of all samples) were excluded from analysis. However, in the lung pieces that were included in the analysis, flow in samples from central areas with large vessels and airways may still have been underestimated because of the weight of the vessels and airways. The results of this methodological artifact would be to decrease the size of the observed hilar-to-peripheral gradient.

Interpretation of the pulmonary blood flow distribution data depends on preservation of lung size and spatial orientation of the lungs in vivo, when foam enveloped in the rigid box. We conserved lung size and shape by carefully opposing the left and right sides in anatomic position and drying the lungs at total lung capacity. Some distortion of pulmonary parenchyma was likely when the lungs were kept inflated at total lung capacity with 25 cmH2O (23), but this should have a small influence on the major findings of this study. We ensured visually that the lungs were oriented properly in the rigid box when foam enveloped to assure sectioning of the lungs in true isogravitational planes.

Heterogeneity of pulmonary blood flow. Pulmonary blood flow heterogeneity in this study was low (30%) compared with previous reports in dogs (11, 27) and goats (25). Melsom et al. (25) examined distribution of pulmonary blood flow in horizontal lung slices in four awake goats. They normalized regional flow to the mean flow in each slice, making a direct comparison with our data difficult. They found a mean heterogeneity of 38%, which probably would have increased if the effect of the vertical gradient had been included. Using similar techniques to examine regional blood flow as in our study, Parker et al. (27) observed a mean heterogeneity of 47% in awake chronically catheterized dogs, and Glenny et al. (11) found a mean heterogeneity of 44% in halothane-anesthetized prone dogs. Methodological differences, such as anesthesia and mechanical ventilation (11), size of piece studied (12), and exclusion of lung regions with a predominance of airways (11), are unlikely to account for perceived interspecies differences in the heterogeneity of pulmonary blood flow. Whereas heterogeneity increases with decreasing size of piece studied, as a result of a fractal branching pattern of the pulmonary circulation (12), regions of similar size (1-1.9 cm3) were studied in dogs and sheep.

Each study excludes some pieces for methodological reasons. These exclusions have little effect on the assessed heterogeneity. Inclusion of all lung regions in young sheep in our study increased pulmonary blood blow heterogeneity only slightly, from 30 ± 6 to 33 ± 7%. Also, blood flow heterogeneity in the dog (11, 27) was not affected by percentage of excluded lung regions. A similar heterogeneity was observed when 10% (27) or 13% (11) of all lung regions were excluded.

Heterogeneity in the right apical lobe was increased compared with the rest of the lung. This was due to a bimodal gradient of flow across sagittal planes in five sheep, with a considerable number of low-flow regions. Their location suggested a relationship to bronchial anatomy in the right apical lobe (9), indicating that these low-flow regions could be secondary to regional hypoventilation, resulting in reduced blood flow due to hypoxic pulmonary vasoconstriction.

Vertical gradients. A major impetus to the present work was to assess the importance of the gravitational distribution of pulmonary blood flow. Questions were raised by studies showing large blood flow variability in isogravitational samples of lung tissue (11, 26) and blood flow gradients not easily explained by the interaction of vascular and alveolar pressures (4, 13, 14, 20) as originally described by West et al. (34). Because most of the in situ studies have used anesthetized animals, and anesthetic agents may cause redistribution of pulmonary blood flow (8, 31), the present study measured regional flow in the awake, unanesthetized animal. We found no significant vertical flow gradient in the dimensions (<18.2 cm) examined. Heterogeneity was not significantly different in gravitational and isogravitational planes, indicating that pulmonary structure effectively overrides influences by gravity in the prone position under zone 3 conditions. However, the present study showed that gravity had little influence, by use of statistical correlations of blood flow distributions. The best way to show the influence of gravity is to study the animals in two conditions that differ in the way gravity acts on the lungs. Addition of the supine position would have added power to the conclusion regarding gravitational influences.

Our results confirmed work using similar techniques, in which the distribution of pulmonary blood flow was not dependent on vertical height in anesthetized and mechanically ventilated prone dogs (11) and in awake, standing horses (18). Our results, however, contrast with some other studies that used a different methodology to reconstruct anatomic blood flow distribution from a large number of regional flows (26, 27). A gravitational gradient showing an increase of blood flow of 4-4.7%/cm down the lung has been reported in awake, chronically instrumented dogs (27) and prone, anesthetized, spontaneously ventilated dogs (26). However, methodological differences may account for the variable results. The studies that observed a gravitational gradient (26, 27) analyzed the distribution of microspheres in blood-filled expanded frozen lungs, which introduces an error in calculation of weight-corrected flow and also allows for less expansion of the lungs than in the exsanguinated animal (7). Furthermore, Nicolaysen et al. (26) calculated mean flow per isogravitational plane, which underestimates flow heterogeneity and gives each plane equal weight, regardless of the number of pieces. Interspecies differences may also contribute to the lack of gravitational gradient in the sheep. Lambs have a high resistance to collateral ventilation (32) and use pulmonary vasomotion more to maintain ventilation-perfusion matching. Theoretically, active vasomotion could partly override other factors that influence pulmonary blood flow distribution, such as gravity.

Despite the differences, the results, which all were obtained in prone animals with lungs mainly in zone 3, suggest that gravity exerts a nonsignificant influence on blood flow distribution under these conditions. Although the vertical gradients were not very different from those in the original work by West et al. (34) employing external detection of the uptake of Xe-133 in isolated dog lungs, the fraction of flow variance explained by the gravitational gradient was very small in the present study, in which higher resolution techniques were employed.

Central-to-peripheral gradients. There was significant inverse dependence of flow on distance from the ipsilateral hilus of the lung, with a mean decrease in flow of 4.0%/cm. The hilar-to-peripheral gradient persisted within transverse planes after correction for trends in the caudal-cranial direction; however, it was smaller (mean decrease in flow of 0.7%/cm). Thus most, but not all, of the hilar-to-peripheral gradient was in the caudal-cranial direction.

Similar radial gradients (although by use of different center points) have been observed in dogs (11, 14-16, 27), humans (17), and ponies (20). The radial gradient accounts for 7.6-10.8% of variability in regional blood flow (11, 27). In these studies, the correlations were relative to center of mass of each lung or lobe, whereas in the present study, the correlation is against the hilar position, which is on the edge of both lungs. It is likely that an even higher correlation of pulmonary blood flow with distance from the hilus would have been observed if the entry of each lobar pulmonary artery were chosen as the center point.

Using different methodology (single-photon-emission-computed tomography with radiolabeled-albumin macroaggregates) and center point (lung mass), Hakim et al. (15) observed a remarkably similar central-to-peripheral blood flow gradient in dogs. Blood flow decreased with a factor of 4:1 from center to periphery of the lung, compared with the 1.5-2.8:1 ratio approximated from our data with the use of different methodology. Although the use of single-photon-emission-computed tomography in this context has been criticized because of attenuation and reconstruction artifacts in the lung periphery (3), the demonstration of a gradient in the present study cannot be attributed to a methodological artifact introduced by the weight correction. The hilar-to-peripheral gradient was still observed when pleural pieces with low weight were excluded. In fact, the methodological artifact of the present technique would have increased the size of the hilar-to-peripheral gradient due to underestimation of flow in the central areas. Therefore, the hilar-to-peripheral gradient of pulmonary blood flow is most likely a function of vascular anatomy, due to branching vessels of larger patterns and regional differences in capillary density.

The fraction of flow variance in the awake sheep lung that was attributed to spatial coordinates, identification by lobe, and hilar-to-peripheral distance was ~34%, in contrast to 12% reported in dogs (11). This greater importance of hilar-to-peripheral gradients in the awake sheep is likely due to interspecies differences, although the presence of anesthesia, mechanical ventilation, variation in cardiac output (16), and choice of center point used to calculate the gradient may also contribute. The hilar-to-peripheral distance was found to be the most important determinant of blood flow variance. In contrast, the dorsal-ventral vector did not contribute significantly to the regional variation of pulmonary blood flow in this experiment. Thus gravitational effects on pulmonary blood flow distribution were effectively overridden in the awake, spontaneously breathing sheep.

In summary, we found a significant hilar-to-peripheral pulmonary blood flow gradient, with a decrease of 4.0%/cm distance from the ipsilateral hilus of the lung in prone, awake, spontaneously breathing lambs. This distance explained 20% of blood flow variability. When the three-dimensional spatial location was added, a mean of 34% of flow variability could be accounted for. There was no significant vertical gravitational gradient of pulmonary blood flow in the awake, prone sheep.

ACKNOWLEDGEMENTS

The authors thank Mical Middaugh, Dr. Jeff Parke, Tina Stafki, and Dr. Susan Bernard; Dowon An for technical help; and Karen Rutherford for secretarial assistance.

FOOTNOTES

   This work was supported by National Heart, Lung, and Blood Institute Grants HL-12174, HL-24163, and HL-02507, The Swedish Society of Medicine (Carin Tryggers Minnesfond), and The Swedish Medical Research Council.

1   Consecutive slices in the three planes were 1.2 cm apart.

Address for reprint requests: K. B. Domino, Dept. of Anesthesiology, Box 356540, University of Washington, Seattle, WA 98195.

Received 21 December 1995; accepted in final form 18 October 1996.

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