Regional hypoxic pulmonary vasoconstriction in prone pigs.

Hypoxic pulmonary vasoconstriction (HPV) is known to affect regional pulmonary blood flow distribution. It is unknown whether lungs with well-matched ventilation (V)/perfusion (Q) have regional differences in the HPV response. Five prone pigs were anesthetized and mechanically ventilated (positive end-expiratory pressure = 2 cmH2O). Two hypoxic preconditions [inspired oxygen fraction (FI(O2)) = 0.13] were completed to stabilize the animal's hypoxic response. Regional pulmonary blood Q and V distribution was determined at various FI(O2) (0.21, 0.15, 0.13, 0.11, 0.09) using the fluorescent microsphere technique. Q and V in the lungs were quantified within 2-cm3 lung pieces. Pieces were grouped, or clustered, based on the changes in blood flow when subjected to increasing hypoxia. Unique patterns of Q response to hypoxia were seen within and across animals. The three main patterns (clusters) showed little initial difference in V/Q matching at room air where the mean V/Q range was 0.92-1.06. The clusters were spatially located in cranial, central, and caudal portions of the lung. With decreasing FI(O2), blood flow shifted from the cranial to caudal regions. We determined that pulmonary blood flow changes, caused by HPV, produced distinct response patterns that were seen in similar regions across our prone porcine model.

Distributions of lung ventilation and perfusion in prone and supine humans exposed to hypergravity.

When normal subjects are exposed to hypergravity [5 times normal gravity (5 G)] there is an impaired arterial oxygenation that is less severe in the prone compared with supine posture. We hypothesized that under these conditions the heterogeneities of ventilation and/or perfusion distributions would be less prominent when subjects were prone compared with supine. Expirograms from a combined rebreathing-single breath washout maneuver (Rohdin M, Sundblad P, and Linnarsson D. J Appl Physiol 96: 1470-1477, 2004) were analyzed for vital capacity (VC), phase III slope, and phase IV amplitude, to analyze heterogeneities in ventilation (Ar) and perfusion [CO(2)-to-Ar ratio (CO(2)/Ar)] distribution, respectively. During hypergravity, VC decreased more in the supine than in the prone position (ANOVA, P = 0.02). Phase III slope was more positive for Ar (P = 0.003) and more negative for CO(2)/Ar (P = 0.007) in the supine compared with prone posture at 5 G, in agreement with the notion of a more severe hypergravity-induced ventilation-perfusion mismatch in supine posture. Phase IV amplitude became lower in the supine than in the prone posture for both Ar (P = 0.02) and CO(2)/Ar (P = 0.004) during hypergravity as a result of the more reduced VC in the supine posture. We speculate that results of VC and phase IV amplitude are due to the differences in heart-lung interaction and diaphragm position between postures: a stable position of the heart and diaphragm in prone hypergravity, in contrast to supine in which the weight of the heart and a cephalad shift of the diaphragm compress lung tissue.

Effects of gravity on lung diffusing capacity and cardiac output in prone and supine humans.

Both in normal subjects exposed to hypergravity and in patients with acute respiratory distress syndrome, there are increased hydrostatic pressure gradients down the lung. Also, both conditions show an impaired arterial oxygenation, which is less severe in the prone than in the supine posture. The aim of this study was to use hypergravity to further investigate the mechanisms behind the differences in arterial oxygenation between the prone and the supine posture. Ten healthy subjects were studied in a human centrifuge while exposed to 1 and 5 times normal gravity (1 G, 5 G) in the anterioposterior (supine) and posterioanterior (prone) direction. They performed one rebreathing maneuver after approximately 5 min at each G level and posture. Lung diffusing capacity decreased in hypergravity compared with 1 G (ANOVA, P = 0.002); it decreased by 46% in the supine posture compared with 25% in the prone (P = 0.01 for supine vs. prone). At the same time, functional residual capacity decreased by 33 and 23%, respectively (P < 0.001 for supine vs. prone), and cardiac output by 40 and 31% (P = 0.007 for supine vs. prone), despite an increase in heart rate of 16 and 28% (P < 0.001 for supine vs. prone), respectively. The finding of a more impaired diffusing capacity in the supine posture compared with the prone at 5 G supports our previous observations of more severe arterial hypoxemia in the supine posture during hypergravity. A reduced pulmonary-capillary blood flow and a reduced estimated alveolar volume can explain most of the reduction in diffusing capacity when supine.

Protective effect of prone posture against hypergravity-induced arterial hypoxaemia in humans.

Patients with acute respiratory distress syndrome have increased lung tissue weight and therefore an increased hydrostatic pressure gradient down the lung. Also, they have a better arterial oxygenation in prone (face down) than in supine (face up) posture. We hypothesized that this effect of the direction of gravity also existed in healthy humans, when increased hydrostatic gradients were induced by hypergravity. Ten healthy subjects were studied in a human centrifuge while exposed to 1 or 5 G in anterio-posterior (supine) or posterio-anterior (prone) direction. We measured blood gases using remote-controlled sampling and gas exchange by mass spectrometry. Hypergravity led to marked impairments of arterial oxygenation in both postures and more so in supine posture. At 5 G, the arterial oxygen saturation was 84.6 +/- 1.2 % (mean +/- S.E.M.) in supine and 89.7 +/- 1.4 % in prone posture (P < 0.001 for supine vs. prone). Ventilation and alveolar PO2 were increased at 5 G and did not differ between postures. The alveolar-to-arterial PO2 difference increased at 5 G to 8.0 +/- 0.2 kPa and 6.6 +/- 0.3 kPa in supine and prone postures (P = 0.003). Arterial oxygenation was less impaired in prone during hypergravity due to a better-preserved alveolo-arterial oxygen transport. We speculate that mammals have developed a cardiopulmonary structure that favours function with the gravitational vector in the posterio-anterior direction.

Extending fluorescent microsphere methods for regional organ blood flow to 13 simultaneous colors.

Seven fluorescent microsphere colors can be used in a single experiment to estimate regional blood flow without correcting for spillover of emitted fluorescence. To extend the method to 13 colors, we compared the accuracy of three methods for spillover correction. Fixed wavelength intensities were corrected by matrix inversion, and synchronous scan spectra were corrected by least squares fit of an overdetermined system of linear equations and by least squares fit of a sum of Gaussian and Lorentzian functions. Correction methods were validated in pigs and sheep by simultaneous injections of radioactive microspheres and fluorescent microspheres of 7, 10, and 13 different colors. We induced extreme changes in flow to create regions with low fluorescent signals bound on either side by high fluorescent signals. Blood flow was determined by radioactivity and by fluorescence using both fixed excitation and emission wavelength pairs and synchronous scanning and then corrected for spillover. Correlation between fluorescent intensity and radioactivity were excellent for all three correction methods [R2 = 0.98 +/- 0.02 (mean +/- SD)]. Low-flow regions requiring large spillover correction had systematic errors for some color combinations in all methods. We conclude that for 13 fluorescent colors spillover error can be minimized so that all three correction methods provide accurate estimates of regional blood flow.

Effect of increasing perfluorocarbon dose on VA/Q distribution during partial liquid ventilation in acute lung injury.

BACKGROUND: Although gas exchange during partial liquid ventilation (PLV) depends on perfluorocarbon liquid, the effect of perfluorocarbon dose on the ventilation-perfusion (VA/Q) distribution is not known. This study investigated how VA/Q distribution of an acutely injured lung is affected during PLV at increasing perfluorocarbon dose. METHODS: In eight rabbits (3.2 +/- 0.1 kg), acute lung injury (ALI) was created by repeated saline lavage (arterial oxygen partial pressure/fraction of inspired oxygen, 37 +/- 11 mm Hg). Three different doses of perfluorodecalin (9 ml/kg = low dose; 13.5 ml/kg = medium dose; 18 ml/kg = functional residual capacity [FRC] dose) were applied in random order during PLV. VA/Q distribution at different doses was evaluated by multiple inert gas elimination technique. RESULTS: Inert gas shunt (63 +/- 21% at ALI) decreased with increasing perfluorocarbon dose (43 +/- 21% at low dose, 29 +/- 10% at medium dose, 11 +/- 9% at FRC dose; P = 0.022). Compared with ALI (0%), the proportion of low VA/Q units was higher at all tested doses (19 +/- 10, 25 +/- 12, and 34 +/- 18%, respectively; all P < 0.05). Compared with ALI (27 +/- 14%), the proportion of normal VA/Q units was not increased at low or medium doses but was increased only at the FRC dose (45 +/- 13%; P = 0.027). CONCLUSIONS: With increasing perfluorocarbon dose during PLV, shunt was reduced from a small dose. The majority shunt units were converted to units showing low VA/Q ratios rather than normal VA/Q ratios. The presence of considerable amount of low VA/Q units across the varying doses of perfluorocarbon suggested that additional measures are necessary during PLV to augment its effect on gas exchange.

Endotoxemia increases relative perfusion to dorsal-caudal lung regions.

Changes in the spatial distribution of perfusion during acute lung injury and their impact on gas exchange are poorly understood. We tested whether endotoxemia caused topographical differences in perfusion and whether these differences caused meaningful changes in regional ventilation-to-perfusion ratios and gas exchange. Regional ventilation and perfusion were measured in anesthetized, mechanically ventilated pigs in the prone position before and during endotoxemia with the use of aerosolized and intravenous fluorescent microspheres. On average, relative perfusion halved in ventral and cranial lung regions, doubled in caudal lung regions, and increased 1.5-fold in dorsal lung regions during endotoxemia. In contrast, there were no topographical differences in perfusion before endotoxemia and no topographical differences in ventilation at any time point. Consequently, endotoxemia increased regional ventilation-to-perfusion ratios in the caudal-to-cranial and dorsal-to-ventral directions, resulting in end-capillary PO2 values that were significantly lower in dorsal-caudal than ventral-cranial regions. We conclude that there are topographical differences in the pulmonary vascular response to endotoxin that may have important consequences for gas exchange in acute lung injury.

Spatial distribution of nutritive and nonnutritive vascular routes in perfused rat hindlimb muscle using microspheres.

Skeletal muscle appears to have two vascular flow routes, nutritive and nonnutritive, where the balance of flow is controlled by vasoconstrictors. In the present study, spatial distributions of the two flow routes in muscles of the perfused rat hindlimb were attempted using fluorescent microspheres (15 microm in diameter). Microspheres were injected during steady-state perfusion with norepinephrine (proposed recruiter of nutritive flow), serotonin (proposed recruiter of nonnutritive flow), or vehicle. The three-dimensional location of individual microspheres in representative muscles was determined using a Fluorescent Imaging CryoMicrotome. Norepinephrine and serotonin each increased perfusion pressure (P < 0.05) but stimulated and inhibited oxygen consumption (P < 0.05), respectively. The distribution of microspheres lodged in muscle was independent of the agent used. Spatial perfusion indices for norepinephrine, serotonin, and vehicle did not differ from each other. Similarly, there was no difference in these indices for a theoretical distribution where microspheres were deliberately positioned in muscle bundle capillaries or interfibrillar connective tissue vessels. We conclude that the nutritive and nonnutritive flow routes are distributed throughout muscle sections consistent with their locations in muscle bundle capillaries and interfibrillar connective tissue, respectively.

Vasomotor tone does not affect perfusion heterogeneity and gas exchange in normal primate lungs during normoxia.

To determine whether vasoregulation is an important cause of pulmonary perfusion heterogeneity, we measured regional blood flow and gas exchange before and after giving prostacyclin (PGI(2)) to baboons. Four animals were anesthetized with ketamine and mechanically ventilated. Fluorescent microspheres were used to mark regional perfusion before and after PGI(2) infusion. The lungs were subsequently excised, dried inflated, and diced into approximately 2-cm(3) pieces (n = 1,208-1,629 per animal) with the spatial coordinates recorded for each piece. Blood flow to each piece was determined for each condition from the fluorescent signals. Blood flow heterogeneity did not change with PGI(2) infusion. Two other measures of spatial blood flow distribution, the fractal dimension and the spatial correlation, did not change with PGI(2) infusion. Alveolar-arterial O(2) differences did not change with PGI(2) infusion. We conclude that, in normal primate lungs during normoxia, vasomotor tone is not a significant cause of perfusion heterogeneity. Despite the heterogeneous distribution of blood flow, active regulation of regional perfusion is not required for efficient gas exchange.

High spatial resolution measurements of organ blood flow in small laboratory animals.

With the use of a newly developed Imaging Cryomicrotome to determine the spatial location of fluorescent microspheres in organs, we validate and report our processing algorithms for measuring regional blood flow in small laboratory animals. Microspheres (15-microm diameter) of four different fluorescent colors and one radioactive label were simultaneously injected into the left ventricle of a pig. The heart and kidneys were dissected, and the numbers of fluorescent and radioactive microspheres were determined in 10 randomly selected pieces. All microsphere counts fell well within the 95% expected confidence limits as determined from the radioactive counts. Fluorescent microspheres (15-microm diameter) of four different colors were also injected into the tail vein of a rat and the left ventricle of a rabbit. After correction for Poisson noise, correlation coefficients between the colors were 0.99 +/- 0.02 (means +/- SD) for the rabbit heart and 0.99 +/- 0.02 for the rat lung. Mathematical dissection algorithms, statistics to analyze the spatial data, and methods to visualize blood flow distributions in small animal organs are presented.

Adaptation of respiratory muscle perfusion during exercise to chronically elevated ventilatory work.

Pneumonectomy (PNX) leads to chronic asymmetric ventilatory loading of respiratory muscles (RM). We measured RM energy requirements during exercise from RM blood flow (Q) using a fluorescent microsphere technique in dogs that had undergone right PNX as adults (adult R-PNX) or as puppies (puppy R-PNX), compared with dogs subjected to right thoracotomy without PNX as puppies (Sham) and to left PNX as adults (adult L-PNX). Ventilatory work (W) was measured during exercise. RM weight was determined post mortem. After adult and puppy R-PNX, the right hemidiaphragm becomes grossly distorted, but W and right costal muscle mass increased only after adult R-PNX. After adult L-PNX, the diaphragm was undistorted; W and left hemidiaphragm RM Q were elevated, but muscle mass did not increase. Mass of parasternal muscle did not increase after adult R-PNX, despite increased Q. Thus muscle mass increased only in response to the combination of chronic stretch and dynamic loading. There was a dorsal-to-ventral gradient of increasing Q within the diaphragm, but the distribution was unaffected by anatomic distortion, hypertrophy, or workload, suggesting a fixed pattern of neural activation. The diaphragm and parasternals were the primary muscles compensating for the asymmetric loading from PNX.

Effects of inertial load and countermeasures on the distribution of pulmonary blood flow.

We assessed the influence of cranial-to-caudal inertial force (+G(z)) and the countermeasures of anti-G suit and positive pressure breathing during G (PBG), specifically during +G(z), on regional pulmonary blood flow distribution. Unanesthetized swine were exposed randomly to 0 G(z) (resting), +3 G(z), +6 G(z), and +9 G(z), with and without anti-G suit and PBG with the use of the Air Force Research Laboratory centrifuge at Brooks Air Force Base (the gravitational force of the Earth, that is, the dorsal-to-ventral inertial force, was present for all runs). Fluorescent microspheres were injected into the pulmonary vasculature as a marker of regional pulmonary blood flow. Lungs were excised, dried, and diced into approximately 2-cm(3) pieces, and the fluorescence of each piece was measured. As +G(z) was increased from 0 to +3 G(z), blood flow shifted from cranial and hilar regions toward caudal and peripheral regions of the lung. This redistribution shifted back toward cranial and hilar regions as anti-G suit inflation pressure increased at +6 and +9 G(z). Perfusion heterogeneity increased with +G(z) stress and decreased at the higher anti-G suit pressures. The distribution of pulmonary blood flow was not affected by PBG. ANOVA indicated anatomic structure as the major determinant of pulmonary blood flow.

Hemodynamic effects of 15-microm-diameter microspheres on the rat pulmonary circulation.

The microsphere method has been used extensively to measure regional blood flow in large laboratory animals. A fundamental premise of the method is that microspheres do not alter regional flow or vascular tone. Whereas this assumption is accepted in large animals, it may not be valid in the pulmonary circulation of smaller animals. Three studies were performed to determine the hemodynamic effects of microspheres on the rat pulmonary circulation. Increasing numbers of 15-microm-diameter microspheres were injected into a fully dilated, isolated-lung preparation. Vascular resistance increased 0.8% for every 100,000 microspheres injected. Microspheres were also injected into an isolated-lung preparation in which vascular tone was increased with hypoxia. Microspheres did not induce vasodilatation, as reported in other vascular beds. Fluorescent microspheres were injected via tail veins into awake rats, and the spatial locations of the microspheres were determined. Regional distributions remained highly correlated when microspheres of one color were injected after microspheres of another color. This indicates that the initial injection did not alter regional perfusion. We conclude that, when used in appropriate numbers, 15-microm-diameter microspheres do not alter regional flow or vascular tone in the rat pulmonary circulation.

Pulmonary blood flow remains fractal down to the level of gas exchange.

The spatial distribution of pulmonary blood flow is increasingly heterogeneous as progressively smaller lung regions are examined. To determine the extent of perfusion heterogeneity at the level of gas exchange, we studied blood flow distributions in rat lungs by using an imaging cryomicrotome. Approximately 150,000 fluorescent 15-microm-diameter microspheres were injected into tail veins of five awake rats. The rats were heavily anesthetized; the lungs were removed, filled with an optimal cutting tissue compound, and frozen; and the spatial location of every microsphere was determined. The data were mathematically dissected with the use of an unbiased random sampling method. The coefficients of variation of microsphere distributions were determined at varying sampling volumes. Perfusion heterogeneity increased linearly on a log-log plot of coefficient of variation vs. volume, down to the smallest sampling size of 0.53 mm(3). The average fractal dimension, a scale-independent measure of perfusion distribution, was 1.2. This value is similar to that of other larger species such as dogs, pigs, and horses. Pulmonary perfusion heterogeneity increases continuously and remains fractal down to the acinar level. Despite the large degree of perfusion heterogeneity at the acinar level, gases are efficiently exchanged.

Selected contribution: measuring the response time of pulmonary capillary recruitment to sudden flow changes.

To determine how rapidly pulmonary capillaries recruit after sudden changes in blood flow, we used an isolated canine lung lobe perfused by two pumps running in parallel. When one pump was turned off, flow was rapidly halved; when it was turned on again, flow immediately doubled. We recorded pulmonary capillary recruitment in subpleural alveoli using videomicroscopy to measure how rapidly the capillaries reached a new steady state after these step changes in blood flow. When flow was doubled, capillary recruitment reached steady state in <4 s. When flow was halved, steady state was reached in approximately 8 s. We conclude that the pulmonary microcirculation responds rapidly to step changes in flow, even in the capillaries that are most distant from the hilum.

Selected contribution: redistribution of pulmonary perfusion during weightlessness and increased gravity.

To compare the relative contributions of gravity and vascular structure to the distribution of pulmonary blood flow, we flew with pigs on the National Aeronautics and Space Administration KC-135 aircraft. A series of parabolas created alternating weightlessness and 1.8-G conditions. Fluorescent microspheres of varying colors were injected into the pulmonary circulation to mark regional blood flow during different postural and gravitational conditions. The lungs were subsequently removed, air dried, and sectioned into approximately 2 cm(3) pieces. Flow to each piece was determined for the different conditions. Perfusion heterogeneity did not change significantly during weightlessness compared with normal and increased gravitational forces. Regional blood flow to each lung piece changed little despite alterations in posture and gravitational forces. With the use of multiple stepwise linear regression, the contributions of gravity and vascular structure to regional perfusion were separated. We conclude that both gravity and the geometry of the pulmonary vascular tree influence regional pulmonary blood flow. However, the structure of the vascular tree is the primary determinant of regional perfusion in these animals.

Correlation between ventilation and perfusion determines VA/Q heterogeneity in endotoxemia.

Endotoxin increases ventilation-to-perfusion ratio (VA/Q) heterogeneity in the lung, but the precise changes in alveolar ventilation (VA) and perfusion that lead to VA/Q heterogeneity are unknown. The purpose of this study was to determine how endotoxin affects the distributions of ventilation and perfusion and the impact of these changes on VA/Q heterogeneity. Seven anesthetized, mechanically ventilated juvenile pigs were given E. coli endotoxin intravenously, and regional ventilation and perfusion were measured simultaneously by using aerosolized and injected fluorescent microspheres. Endotoxemia significantly decreased the correlation between regional ventilation and perfusion, increased perfusion heterogeneity, and redistributed perfusion between lung regions. In contrast, ventilation heterogeneity did not change, and redistribution of ventilation was modest. The decrease in correlation between regional ventilation and perfusion was responsible for significantly more VA/Q heterogeneity than were changes in ventilation or perfusion heterogeneity. We conclude that VA/Q heterogeneity increases during endotoxemia primarily as a result of the decrease in correlation between regional ventilation and perfusion, which is in turn determined primarily by changes in perfusion.

Exercise alters fractal dimension and spatial correlation of pulmonary blood flow in the horse.

We determined the changes in fractal dimensions and spatial correlations of regional pulmonary blood flow with increasing exercise in race horses (n = 4) by using 15-microm fluorescent microspheres. Fluorescence was measured to quantitate regional blood to 1.3-cm(3) samples (n = 1,621-2,503). Perfusion distributions were characterized with fractal dimensions (a measure of spatial variability) and spatial correlations. On average, the fractal dimension decreased with exercise (trot 1.216 to gallop 1.173; P < 0. 05) despite a variable fractal dimension at rest. Spatial correlation of flow to neighboring pieces increased with exercise (trot 0.57 +/- 0.074 to gallop 0.73 +/- 0.051) and was inversely correlated with fractal dimension, indicating better spatial correlation as blood flow distribution becomes more uniform. This is the first study to document a change in fractal dimension as a result of increasing pulmonary blood flow. Spatial differences in response to vasoregulatory mediators may play a role in this phenomenon.

Fractal nature of regional ventilation distribution.

High-resolution measurements of pulmonary perfusion reveal substantial spatial heterogeneity that is fractally distributed. This observation led to the hypothesis that the vascular tree is the principal determinant of regional blood flow. Recent studies using aerosol deposition show similar ventilation heterogeneity that is closely correlated with perfusion. We hypothesize that ventilation has fractal characteristics similar to blood flow. We measured regional ventilation and perfusion with aerosolized and injected fluorescent microspheres in six anesthetized, mechanically ventilated pigs in both prone and supine postures. Adjacent regions were clustered into progressively larger groups. Coefficients of variation were calculated for each cluster size to determine fractal dimensions. At the smallest size lung piece, local ventilation and perfusion are highly correlated, with no significant difference between ventilation and perfusion heterogeneity. On average, the fractal dimension of ventilation is 1.16 in the prone posture and 1. 09 in the supine posture. Ventilation has fractal properties similar to perfusion. Efficient gas exchange is preserved, despite ventilation and perfusion heterogeneity, through close correlation. One potential explanation is the similar geometry of bronchial and vascular structures.