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.     

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.     

The spatial-temporal redistribution of pulmonary blood flow with postnatal growth.

The pulmonary vascular tree undergoes remarkable postnatal development and remodeling. While a number of studies have characterized longitudinal changes in vascular function with growth, none have explored regional patterns of vascular remodeling. We therefore studied six neonatal pigs to see how regional blood flow changes with growth. We selected pigs because of their rapid growth and their similarities to human development with respect to the pulmonary vascular tree. Fluorescent microspheres of varying colors were injected into the pulmonary circulation to mark regional blood on days 3, 12, 27, 43, and 71 after birth. The animals were awake and in the prone posture for all injections. The lungs were subsequently removed, air dried, and sectioned into approximately 2-cm(3) pieces. Flow on each injection day was determined for each piece. Despite the increase in the hydrostatic gradient in the lung with growth, there was a strong correlation between blood flow to the same lung piece when compared on days 3 and 71 (0.73 +/- 0.12). Although a dorsal-ventral gradient of perfusion did not exist on day 3, blood flow increased more in the dorsal region by day 12 and then gradually became more uniform by day 71. Although most of the lung pieces did not show any discernable pattern of blood flow redistribution, there were spatial patterns of blood flow redistribution that were similar across animals. Our findings suggest that local mechanisms, shared across animals, guide regional changes in vascular resistance or vasoregulation during postnatal development. In the pig, these mechanisms act to produce more uniform flow in the normal posture for an ambulating quadruped. The stimuli for these changes have not yet been identified.     

Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation.

Vascular infusions of 15-microm-diameter microspheres are used to study pulmonary blood flow distribution. The sites of microsphere lodging and their effects on microvascular perfusion are debated but unknown. Using intravital microscopy of the subpleural surface of rat lungs, we directly observed deposition of fluorescent microspheres. In a pump-perfused lung model, approximately 0.5 million microspheres were infused over 30 s into the pulmonary artery of seven rats. Microsphere lodging was analyzed for the location in the microvasculature and the effect on local flow after lodging. On average, we observed 3.2 microspheres per 160 alveolar facets. The microspheres always entered the arterioles as singlets and lodged at the inlets to capillaries, either in alveolar corner vessels or small arterioles. In all cases, blood flow continued either around the microspheres or into the capillaries via adjacent pathways. We conclude that 15-microm-diameter microspheres, in doses in excess of those used in typical studies, have no significant impact on pulmonary capillary blood flow distribution.     

Actions of opiate agonists, naloxone, and paraben preservatives in the rat lung circulation

Previous studies have documented direct vascular effects of opiate substances in the systemic circulation. Because opiate receptors have been identified in the lung, we wondered whether opiate substances might affect vasoreactivity in the lung circulation. We studied the pulmonary vascular effects of three opiate agonists: morphine, leucine-enkephalin, and dynorphin, as well as the opiate receptor antagonist naloxone, in isolated rat lungs perfused with a cell- and plasma-free salt solution. Because of previous reports of the smooth muscle effects of the methyl- and propylparaben preservatives in the naloxone preparation, we also studied the pulmonary vascular effects of these preservatives in the rat lung circulation. We found that morphine, a mu-receptor agonist, leucine-enkephalin, a delta-receptor agonist, and dynorphin, a kappa-receptor agonist, caused no immediate vascular effect when injected into the pulmonary artery. In addition, morphine did not affect the pulmonary vasoconstrictions induced by hypoxia, angiotensin II, or potassium chloride. The commercial preparation of naloxone, Narcan, caused a marked vasodilation during hypoxic pulmonary vasoconstriction. However, this effect was entirely attributable to the preservatives methyl- and propylparaben, as pure naloxone had no effect on either the baseline pulmonary vascular tone or the vasoconstrictive response to hypoxia. We conclude that opiate receptor agonists and antagonists do not affect vasoreactivity in the rat lung circulation and that the methyl- and propylparaben preservatives in Narcan are pulmonary vasodilators.     

Changes of regional blood flow induced by atrial natriuretic factor (ANF) in conscious rats

The effect of synthetic atrial natriuretic factor (ANF 101-126) has been studied on regional blood flow distribution. Microspheres (15 +/- 3 microns), labelled with either 113Sn or 57Co, were injected through an intraventricular cannula into conscious rats while a reference blood sample was withdrawn. Two minutes after the first microspheres injection either ANF or NaCl were injected. Five minutes later, the second microspheres injection was administered, and after two minutes the animals were sacrificed, and several tissues removed and counted. Percent of flow distribution, cardiac output and tissue blood flow were calculated by standard formulas. ANF produced a significant increase in absolute blood flow in lungs, heart, spleen, kidneys and testes. Total renal blood flow and total splanchnic blood flow were also increased in ANF-injected animals. No significant changes were observed in cardiac output. It is suggested that the natriuretic and hypotensive responses to ANF in vivo may be, at least partially, explained by its hemodynamic effects.     

Measurement of the blood flow in various areas of the rat brain by means of microspheres]

A method is described to measure regional blood flow in different structures of the rat brain. Microspheres (15 micron) are injected, the brain is sectioned, stained for myeline, radioautographs are prepared and the microspheres in the different structures are counted. The values obtained for different brain structures are counted. The values obtained for different brain regions (cortex, corpus callosum, thalamus hipocampus, hypothalamic region, colliculi, cerebellum, pons, medulla) compare well with those published by others on larger animals. In rats fed 1% of lead from birth, higher blood flow is found in the cortex and a lower one in the interior part of the brain compared to controls.     

Thromboxane receptor analog, U-46619, redistributes pulmonary microvascular perfusion in isolated rat lungs.

Effects of vasoconstriction on the distribution of perfusion among alveoli are not well understood. To address this, we used a new method we developed to determine how microvascular perfusion distribution was affected by a potent vasoconstrictor, the thromboxane receptor analog U-46619. Our method was to infuse 4-microm-diameter fluorescent latex microspheres into the circulation of isolated rat lungs vasoconstricted with U-46619. We used a confocal microscope to image trapping patterns of the particles in dried sections of the lungs and then used dispersion index analysis to quantify the particle patterns in the images, which encompassed approximately 2,000 alveoli. Dispersion indexes revealed significantly more particle clustering (inhomogeneous distribution) in vasoconstricted lungs than in normal flow controls or in controls in which flow was reduced by either lowering pulmonary arterial pressure or raising left atrial pressure. These results suggest that vasoconstriction occurred in the microvessels themselves, which are much smaller vessels than those previously thought to be capable of vasoconstriction.     

Differential uptake of endothelin-1 by the coronary and pulmonary circulations.

Substantial removal of the vasoconstrictor peptide endothelin-1 (ET-1) by the pulmonary circulation has been reported to occur in perfused guinea pig and rat lungs. We examined the uptake of ET-1 by coronary and pulmonary circulations of the rabbit by measuring single-pass extraction of ET-1 in the isolated heart and lung. In separate experiments, each organ was perfused at 30 ml/min with Krebs-albumin (3%) solution. A bolus of 125I-ET-1 and [14C]dextran in 0.3 ml Krebs-albumin solution was injected, and extraction of endothelin (EET), relative to that of an intravascular reference indicator, [14C]dextran, was determined by multiple indicator-dilution technique. EET was 5 +/- 2% (SE) in the heart and 49 +/- 4% in the lung. Increasing flow rate in the lung preparation to approximate the mean transit time in the heart preparation did not significantly alter EET. Despite insignificant uptake of ET-1, the coronary circulation extracted an angiotensin-converting enzyme inhibitor (351A) and metabolized a synthetic angiotensin-converting enzyme substrate (benzoyl-phenyl-alanyl-proline), both properties of the normal pulmonary circulation. We therefore conclude that there is no significant ET-1 uptake in the coronary vascular bed.     

Hemodynamic effects of postpulmonary administration of prostaglandin D2 in fetal animals

Dose-response relationships in pulmonary vascular resistance (PVR), mean systemic arterial pressure (SAP), and heart rate (HR) to left atrial administration of prostaglandin D2 (PGD2) were determined in five fetal lambs. Fetuses were delivered by cesarean section from chloralose anesthetized ewes with the umbilical circulation maintained intact. Fetuses were prevented from breathing thus maintaining pulmonary vascular tone in the elevated fetal state. Blood was withdrawn from the inferior vena cava and pumped at constant flow into the lower left lobe of the fetal lung. Postpulmonary infusions of PGD2 brought about dose-dependent decreases in pulmonary vascular resistance. Heart rate tended to increase in fetal lambs. Mean systemic arterial pressure increased in the fetal lambs at all doses tested except for the largest dose (44.14 micrograms/kg X min), which produced slight hypotension. These data demonstrate that exposure to the systemic circulation prior to entering the pulmonary vasculature does not alter the preferential dilator action of PGD2 on fetal pulmonary vessels nor does it produce significant systemic hypotension.     

Cardiovascular response to histamine during normoxaemia and hypoxaemia in piglets

The cardiovascular effects of exogenously administered histamine were investigated in conscious newborn piglets aged 10-11 days during normoxia (21% (v/v) O2) and during isocapneic alveolar hypoxia (10% O2, 3% CO2, 87% N2) to determine its influence on preexisting vascular tone. In the first set of experiments (n = 6), four histamine doses (1,10,50,100 micrograms/kg) were tested in sequence during normoxia. Histamine was injected intravenously and cardiovascular variables were recorded. Heart rate increased at all doses studied. Pulmonary and systemic arterial pressures, cardiac output and stroke volume were unchanged at the low histamine doses (1 and 10 micrograms), but all decreased at the high doses (50 and 100 micrograms). Pulmonary and systemic vascular resistances were unchanged at each dose. In the second set of experiments (n = 7), two histamine doses (1 and 5 micrograms/kg) were administered during alveolar hypoxia. Hypoxia caused increases in heart rate and pulmonary arterial pressure and resistance. After injection of each dose of histamine, pulmonary pressure and resistance decreased but remained higher than baseline. No other measured cardiovascular variables were altered. Thus, during normoxia histamine did not alter vascular tone, but high doses did adversely affect myocardial function. During alveolar hypoxia histamine caused weak pulmonary vasodilation at doses that did not alter systemic vascular tone. Histamine is not a potent modifier of the circulation in the newborn piglet during conditions of normoxaemia or hypoxaemia.     

Pulmonary vascular effects of bombesin and gastrin-releasing peptide in conscious newborn lambs

A growing body of anatomic findings has led investigators to suggest that peptide-containing pulmonary neuroendocrine cells may control pulmonary vascular tone. One such peptide, bombesin, has been found in increased quantities in the lungs of infants with pulmonary disorders that cause pulmonary hypertension. Therefore we studied the effect on pulmonary vascular tone of bombesin, and its C-terminal analog, gastrin-releasing peptide (GRP). Normoxic and hypoxic unsedated newborn lambs with chronically implanted flow probes around the right and left pulmonary arteries were used. Bombesin and GRP were injected into one pulmonary artery only, and direct effects of these peptides were determined by comparing the flow changes in the injected vs. the uninjected lung. Bombesin had no measurable effect on pulmonary vascular resistance under any condition or at any dose (0.1-60 micrograms/kg). Systemic blood pressure increased significantly (12%) after a 10-micrograms/kg dose. GRP was devoid of any measurable hemodynamic effects, even at a dose of 10 micrograms/kg. If pulmonary neuroendocrine cells help regulate vascular resistance, bombesin does not appear to play a role.     

Pulmonary vascular effects of Leu5-enkephalin in conscious newborn lambs

Anatomic evidence suggests that leu5-enkephalin (Leu5-enk) may be involved in the physiologic control of pulmonary vascular tone. Information regarding its pulmonary vascular effect is limited; we therefore studied its effect on the immature pulmonary circulation. Normoxic and hypoxic unsedated newborn lambs with chronically implanted flow probes around the right and left pulmonary arteries were used. Leu5-enk was injected into one pulmonary artery only, so that any direct effect of the peptide on the pulmonary vessels could be determined by measuring changes in the ratio of blood flow to the injected versus the non-injected lung. Leu5-enk caused a small but significant increase in pulmonary artery pressure without increasing cardiac output or left atrial pressure (threshold = 1 microgram/kg); it is therefore a pulmonary vasoconstrictor. At a dose of 10 micrograms/kg, Leu5-enk also raised pulmonary artery pressure (20.6 mmHg to 23.9 mmHg; F(8,36) = 15.1 p less than 0.001) and calculated PAR (14.6 to 16.1 units; NS). However, the ratio of blood flow to the two lungs did not change; thus, Leu5-enk does not appear to directly act on pulmonary vessels, but rather through an intermediary to produce pulmonary vasoconstriction. This indirect pulmonary vasoconstriction was blocked by pretreatment with naloxone (3 mg/kg). We conclude that Leu5-enk is a pulmonary vasoconstrictor, albeit a weak one, in the lamb and may therefore play a role in pulmonary vascular homeostasis. This vasoconstriction does not seem to be due to a direct effect on pulmonary vessels by Leu5-enk, but may be effected through a neural or hormonal intermediary.     

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.     

Evaluation of the size of the microsphere loss from the myocardium in rats in a chronic experiment

The loss of radioactive microspheres (15 microns in diameter) from the rat myocardium was investigated during chronic experiment. Microspheres were injected to donor rats, the hearts were removed 20-30 min later and the number of microspheres trapped in the heart was determined on gamma-counter. After that the hearts were transplanted into the abdominal cavity of recipient rats using microsurgery technique. 5-24 days later the recipients were killed, the transplanted hearts were removed and radioactivity was measured again. It was shown that not more than 10% of microspheres were lost from the myocardium over a 5-24-day period, with the value of microsphere loss independent of the time period.     

Effects of nociceptive stimuli on the pulmonary circulation in the ovine fetus

The fetus is able to exhibit a stress response to painful events, and stress hormones have been shown to modulate pulmonary vascular tone. At birth, the increased level of stress hormones plays a significant role in the adaptation to postnatal life. We therefore hypothesized that pain may alter pulmonary circulation in the perinatal period. The hemodynamic response to subcutaneous injection of formalin, which is used in experimental studies as nociceptive stimulus, was evaluated in chronically prepared, fetal lambs. Fetal lambs were operated on at 128 days gestation. Catheters were placed into the ascending aorta, superior vena cava, and main pulmonary artery. An ultrasonic flow transducer was placed around the left pulmonary artery. Three subcutaneous catheters were placed in the lambs' limb. The hemodynamic responses to subcutaneous injection of formalin, to formalin after fetal analgesia by sufentanil, and to sufentanil alone were recorded. Cortisol and catecholamine concentrations were also measured. Pulmonary vascular resistances (PVR) increased by 42% (P < 0.0001) after formalin injection. Cortisol increased by 54% (P = 0.05). During sufentanil infusion, PVR did not change significantly after formalin. Cortisol increased by 56% (P < 0.05). PVR did not change during sufentanil infusion. Norepinephrine levels did not change during any of the protocols. Our results indicate that nociceptive stimuli may increase the pulmonary vascular tone. This response is not mediated by an increase in circulating catecholamine levels. Analgesia prevents this effect. We speculate that this pulmonary vascular response to nociceptive stimulation may explain some hypoxemic events observed in newborn infants during painful intensive care procedures.     

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.     

Acute hypoxia alters eicosanoid production of perfused pulmonary artery endothelial cells in culture.

Hypoxia alters vascular tone which regulates regional blood flow in the pulmonary circulation. Endothelial derived eicosanoids alter vascular tone and blood flow and have been implicated as modulators of hypoxic pulmonary vasoconstriction. Eicosanoid production was measured in cultured bovine pulmonary endothelial cells during constant flow and pressure perfusion at two oxygen tensions (hypoxia: 4% O2, 5% CO2, 91% N2; normoxia: 21% O2, 5% CO2, 74% N2). Endothelial cells were grown to confluence on microcarrier beads. Cell cartridges (N = 8) containing 2 ml of microcarrier beads (congruent to 5 x 10(6) cells) were constantly perfused (3 ml/min) with Krebs' solutions (pH 7.4, T 37 degrees C) equilibrated with each gas mixture. After a ten minute equilibration period, lipids were extracted (C18 Sep Pak) from twenty minute aliquots of perfusate over three hours (nine aliquots per cartridge). Eicosanoids (6-keto PGF1 alpha; TXB2; and total leukotriene [LT - LTC4, LTD4, LTE4, LTF4]) were assayed by radioimmunoassay. Eicosanoid production did not vary over time. 6-keto PGF1 alpha production was increased during hypoxia (normoxia 291 +/- 27 vs hypoxia 395 +/- 35 ng/min/gm protein; p less than 0.01). Thromboxane production (normoxia 19 +/- 2 vs hypoxia 20 +/- 2 ng/min/gm protein) and total leukotriene production (normoxia 363 +/- 35 vs hypoxia 329 +/- 29 ng/min/gm protein) did not change with hypoxia. These data demonstrated that oxygen increased endothelial prostacyclin production but did not effect thromboxane or leukotriene production.     

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

The role of endothelium-derived hyperpolarizing factor (EDHF) in regulating the pulmonary circulation and the participation of cytochrome P-450 (CYP450) activity and gap junction intercellular communication in EDHF-mediated pulmonary vasodilation are unclear. We tested whether tonic EDHF activity regulated pulmonary vascular tone and examined the mechanism of EDHF-mediated pulmonary vasodilation induced by thapsigargin in salt solution-perfused normotensive and hypoxia-induced hypertensive rat lungs. After blockade of both cyclooxygenase and nitric oxide synthase, inhibition of EDHF with charybdotoxin plus apamin did not affect either normotensive or hypertensive vascular tone or acute hypoxic vasoconstriction but abolished thapsigargin vasodilation in both groups of lungs. The CYP450 inhibitors 7-ethoxyresorufin and sulfaphenazole and the gap junction inhibitor palmitoleic acid, but not 18alpha-glycyrrhetinic acid, inhibited thapsigargin vasodilation in normotensive lungs. None of these agents inhibited the vasodilation in hypertensive lungs. Thus tonic EDHF activity does not regulate either normotensive or hypertensive pulmonary vascular tone or acute hypoxic vasoconstriction. Whereas thapsigargin-induced EDHF-mediated vasodilation in normotensive rat lungs involves CYP450 activity and might act through gap junctions, the mechanism of vasodilation is apparently different in hypertensive lungs.     

Effect of cardiac output on gravity-dependent and nondependent inequality in pulmonary blood flow

To examine the effect of cardiac output (CO) on the gravity-nondependent distribution of pulmonary blood flow, 2 X 10(6) 99mTc-labeled albumin microspheres (20 microns) were injected at end expiration into dogs (anesthetized, supine, and breathing room air spontaneously). Two animals were injected at their resting CO, two were injected during increased CO (arteriovenous fistulas induced), and two were injected at low CO (phlebotomy induced). The chest was opened and the lungs were removed, drained of blood, and dried while fully inflated. Single-photon emission-computed tomography was performed on the dry lungs to map the distribution of activity in transverse, coronal, and sagittal slices. The results confirmed the presence of a central-peripheral gravity-nondependent gradient and showed that increases in CO were associated with increases in absolute flow to both the central and peripheral regions of the lung with persistence of the central-peripheral gradient. These observations were further confirmed by direct imaging of midcoronal slices. Examination of the average flow in vertical and horizontal slices showed that, when zone 1 was not present, changes in CO affected all slices uniformly, such that when the CO doubled, the absolute flow in every slice in all three planes also doubled. We conclude that, with the exception of recruitment and derecruitment of vascular channels in the upper regions of the lung (zone 1), when CO changes, the blood flow everywhere in the lung changes uniformly and in proportion to the CO. This uniform increase in blood flow is consistent with the three-dimensional nature and resistive properties of the pulmonary vascular tree.