Density indicator method to measure pulmonary blood flows

The injection of plasma, saline, or erythrocyte (RBC) concentrate into the pulmonary circulation produces a change in the gravimetric density of the blood outflow similar to the dilution curve of dye. We used an improved density-measuring system to assess the flow of these density indicators through the lung in vivo and in vitro perfused dog lobe. From the in vitro density-dilution curves of plasma and RBC concentrate we calculated the pulmonary flow rate and found it to be 1.04 +/- 0.02 (SD) times the measured one. The outflow-dilution curves of gravimetric density were not as broad as those of optical density following in vivo injection of plasma bolus containing indocyanine green, and the gravimetric measurements dipped to base line, whereas the optical measurement did not. The density-dilution curves of isotonic saline injection are similar to that of plasma. Following injection of RBC concentrates with the dye, density changes in the pulmonary outflow lag behind the emergence of the dye. This was presumably related to RBC aggregation in the concentrates. In reference to the injected plasma, no loss in the density indicators for saline and RBC injection was observed. Based on these results and the similarity of the density indicators to the blood, we conclude that the plasma and isotonic saline are good density indicators to be used for the determination of pulmonary blood flows.     

Pulmonary inflammation alters the lung disposition of lipophilic amine indicators.

Many lipophilic amine compounds are rapidly extracted from the blood on passage through the pulmonary circulation. The extent of their extraction in normal lungs depends on their physical-chemical properties, which affect their degree of ionization, lipophilicity, and propensity for interacting with blood and tissue constituents. The hypothesis of the present study was that changes in the tissue composition that occur during pulmonary inflammation would have a differential effect on the pulmonary extraction of lipophilic amines having different properties. If so, measurement of the extraction patterns for a group of lipophilic amines, having different physical-chemical properties, might provide a means for detecting and identifying lung tissue abnormalities. To evaluate this hypothesis, we measured the pulmonary extraction patterns for four lipophilic amines, [(14)C]diazepam, [(3)H]alfentanil, [(14)C]lidocaine, and [(14)C]codeine, along with two hydrophilic compounds, (3)HOH and [(14)C]phenylethylamine, after the bolus injection of these indicators into the pulmonary artery of isolated lungs from normal rabbits and from rabbits with pulmonary inflammation induced by an intravenous injection of complete Freund's adjuvant. The pulmonary extraction patterns, parameterized using a previously developed mathematical model, were, in fact, differentially altered by the inflammatory response. For example, the tissue sequestration rate, k(seq) (ml/s), per unit (3)HOH accessible extravascular lung water volume significantly increased for diazepam and lidocaine, but not for codeine and alfentanil. The results are consistent with the above hypothesis and suggest the potential for using lipophilic amines as indicators for detection and quantification of changes in lung tissue composition associated with lung injury and disease.     

A compartmental capillary, convolution integration model to investigate nutrient transport and metabolism in vivo from paired indicator/nutrient dilution curves.

Thirty-three paired indicator/nutrient dilution curves across the mammary glands of four cows were obtained after rapid injection of para-aminohippuric acid (PAH) plus glucose into the external iliac artery. For the measurement of extracellular volume and kinetics of nutrient uptake from indicator dilution curves, several models of solute dispersion and disappearance have been proposed. The Crone-Renkin models of exchange in a single capillary assume negligible washout of solutes from the extracellular space and do not describe entire dilution curves. The Goresky models include a distribution of capillary transit times to generate whole system outflow profiles but require two indicators to parametize extracellular behavior. A compartmental capillary, convolution integration model is proposed that uses one indicator to account for the extracellular behavior of the nutrient after a paired indicator/nutrient injection. With the use of an iterative approach to least squares, unique solutions for nonexchanging vessel transit time t(mu) and its variance sigma were obtained from all 33 PAH curves. The average of heterogeneous vascular transit times was approximated as 2sigma = 8.5 s. The remainder of indicator dispersion was considered to be due to washout from a well-mixed compartment representing extracellular space that had an estimated volume of 5.5 liters or 24% of mammary gland weight. More than 99% of the variation in the time course of venous PAH concentration after rapid injection into the arterial supply of the mammary glands was explained in an unbiased manner by partitioning the organ into a heterogeneous nonexchanging vessel subsystem and a well-mixed compartmental capillary subsystem.     

Toluidine blue O and methylene blue as endothelial redox probes in the intact lung

There is increasing evidence that the redox activities of the pulmonary endothelial surface may have important implications for the function of both lungs and blood. Because of the inherent complexity of intact organs, it can be difficult to study these activities in situ. Given the availability of appropriate indicator probes, the multiple-indicator dilution (MID) method is one approach for dealing with some aspects of this complexity. Therefore, the objectives of the present study were to 1) evaluate the potential utility of two thiazine redox indicators, methylene blue (MB) and toluidine blue O (TBO), as MID electron acceptor probes for in situ pulmonary endothelium and 2) develop a mathematical model of the pulmonary disposition of these indicators as a tool for quantifying their reduction on passage through the lungs. Experiments were carried out using isolated rabbit lungs perfused with physiological salt solution with or without plasma albumin over a range of flow rates. A large fraction of the injected TBO disappeared from the perfusate on passage through the lungs. The reduction of its oxidized, strongly polar, relatively hydrophilic blue form to its colorless, highly lipophilic reduced form was revealed by the presence of the reduced form in the venous effluent when plasma albumin was included in the perfusate. MB was also lost from the perfusate, but the fraction was considerably smaller than for TBO. A distributed-in-space-and-time model was developed to estimate the reduction rate parameter, which was approximately 29 and 1.0 ml/s for TBO and MB, respectively, and almost flow rate independent for both indicators. The results suggest the utility particularly of TBO as an electron acceptor probe for MID studies of in situ pulmonary endothelium and of the model for quantitative evaluation of the data.     

Influence of endothelial volume on kinetics of reacting indicators in the lung

The purpose of this work is to show mathematically the relationship between the classical maximum velocity of reaction, Vmax, for enzyme kinetics and an analogous parameter, Vmax, derived by Linehan and Dawson (J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 47:404-411, 1979) for the analysis of tracers which disappear by saturation kinetics from the lung circulation during the passage of indicators after bolus injection. Rederivation of the original equation for the combination of flow and reaction in a capillary showed that Vmax is equal to the product of enzyme Vmax and the volume of endothelium, Ve, in which the enzyme resides. This implies that Vmax interpreted from multiple-indicator curves in the lung by the Linehan-Dawson method is a combination of an enzyme characteristic Vmax and a measure of functioning capillary surface during passage, Ve. Lung injury could change Vmax, functioning surface (Ve), or both.     

Use of diazepam for interpreting changes in extravascular lung water.

Estimates of extravascular lung water volume (Qew) by use of the multiple indicator-dilution method with a hydrophilic indicator such as tritiated water, along with a vascular reference indicator, depend not only on tissue hydration but also on tissue perfusion. Separation of these effects might be facilitated if both hydrophilic and lipophilic indicators were used, with the assumption that the extravascular volume accessible to the lipophilic indicator would be independent of hydration. We found that in isolated perfused dog lung lobes the extravascular volume accessible to the lipophilic amine [14C]diazepam (Qed) was inversely proportional to the albumin concentration of the perfusate. This suggested that while the bolus was in the lungs, only a small fraction of the diazepam was in the aqueous phase of either lung tissue or perfusate. Changing the flow rate over a fairly wide range had little influence on the pattern of the tritiated water or [14C]diazepam effluent concentration curves when time was normalized to the lobar mean transit time. This suggests that the association of the diazepam with both the plasma albumin and the lipoid fraction of the tissue was in very rapid equilibrium on the time scale of a single pass through the lung lobe and that there was little barrier to its diffusion to and from the tissue. When the extravascular water volume was increased by either raising the hydrostatic pressure or instilling saline into the airways, both Qew and Qew/Qed increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

O2 exchange between blood and brain tissues studied with 18O2 indicator-dilution technique

A technique has been developed to record 18O2 dilution curves of an organ in vivo by use of 51Cr-labeled erythrocytes as a reference tracer. The technique employs anaerobic sampling of venous outflow following an intraarterial injection of tracer-laden blood and off-line determination of [18O2] and [51Cr] profiles in the venous outflow. O2 and reference indicator-dilution curves of cerebral circulation were recorded in eight experiments with six halothane-anesthetized dogs. Autologous blood labeled with the tracers was injected into a carotid artery, and brain venous outflow was sampled from the sagittal sinus. The total net extraction of O2 tracer was equal to the extraction of elemental O2. Instantaneous extraction of 18O2 along the outflow curve fell linearly with time, from an initial value of 0.6-0.7 to very small or even negative values toward the end of a pulse. This indicates that O2 undergoes a flow-limited distribution. In all experiments, the mean transit time of unmetabolized 18O2 was longer than the mean transit time of the Cr tracer. An index of the tissue O2 dilution space, hence the mean tissue PO2, is calculated from this data with the use of a modified central volume principle. This estimate of mean tissue PO2 increases as a linear function of sagittal sinus PO2 with a slope of 0.97. The method may provide an index of the critical PO2 of venous blood, the PO2 below which O2 diffusion from blood to tissue may limit its rate of metabolic uptake.     

Influence of vascular distending pressure on regional flows in isolated perfused dog lungs

To confirm the regional differences in vascular pressure vs. flow properties of lung regions that have been documented in zone 2 conditions [pulmonary venous pressure (Ppv) less than alveolar pressure], regional distending pressure vs. flow curves in zone 3 were generated by use of isolated blood-perfused dog lungs (3 right and 5 left lungs). Each lung was kept inflated at constant inflation pressure (approximately 50% of full inflation volume) while radioactively labeled microspheres were injected at different settings of Ppv. To achieve maximal vascular distension, Ppv was increased to approximately 30 cmH2O above alveolar pressure for the first injection. Subsequent injections were made at successively lower Ppv's. The difference between pulmonary arterial pressure and Ppv was kept constant for all injections. As was found in zone 2 conditions, there were differences in the regional distending pressure vs. flow curves among lung regions. To document the regional variability in the curves, the distribution of flow at a regional Ppv of 30 cmH2O above alveolar pressure was analyzed. There was a statistically significant linear gradient in this flow distribution from dorsal to ventral regions of the lungs but no consistent gradient in the caudad to cephalad direction. These results indicate that, even in near-maximally distended vessels, the dorsal regions of isolated perfused dog lungs have lower intrinsic vascular resistance compared with ventral regions.     

Glomerular and postglomerular transcapillary exchange in dog kidney

The multiple-indicator dilution technique (MIDT) was used to study glomerular and postglomerular permselectivity to neutral dextran molecular weight markers in anesthetized dogs undergoing mannitol diuresis. Renal vein and urine outflow curves were obtained after an intraarterial pulse injection of 125I-labeled albumin (plasma reference), creatinine (extracellular reference), [14C]inulin, and chromatographically homogeneous 3H-labeled neutral dextrans. The urine recovery data reflect solute losses across the glomerulus. The renal vein outflow curves contain information about both glomerular and postglomerular extraction. For dextrans less than 13,500 daltons the urine transit pattern was superimposed on the glomerular markers creatinine and inulin. Relative to 125I-labeled albumin, the renal vein recoveries for creatinine, inulin, and dextrans (less than 13,500 daltons) were all equal. The renal vein mean transit time (tdextran) was greater than talbumin and less than tcreatinine. With increasing dextran size, tdextran progressively decreased. Eventually for dextrans greater than 15,500 daltons, tdextran became equal to talbumin in the renal vein, whereas urine recovery relative to inulin decreased with increasing size. Urine recovery of 3H-labeled dextran (ED) relative to inulin (Ei) provided a measure of unidirectional fractional glomerular extraction (ED/Ei). Constant infusion fractional clearance measurement of the same dextrans [U/P)D/(U/P)i) was found to equal ED/Ei obtained from the MIDT. Within the autoregulatory range of glomerular filtration, ED/Ei was invariant with reduction in renal plasma flow. Therefore, under the experimental conditions employed in the present study, diffusion across the glomerulus was negligible relative to convection. This permitted estimation of the reflection coefficients for the series of neutral dextrans. Postglomerular extraction was markedly flow dependent, which implies a major diffusion limitation. Application of a barrier-limited distributed model permitted quantitation of postglomerular capillary permeability coefficients for neutral markers of less than 5000 daltons.     

The Nature of Transcapillary Exchange in the Liver

The nature of transcapillary exchange in the liver was explored by applying the single-injection multiple-indicator dilution technique to the liver. Labelled red cells and a group of passively distributed substances were introduced into the portal vein in the dog. The diffusible substances were delayed during their passage through the liver and the outflow pattern produced by the delay was found, in each case, to correspond to that situation in which the diffusion of these substances across the sinusoidal wall was so rapid in relation to flow that it could be considered complete at every point along the length, a situation which may be termed flow-limited. The calculated volume of distribution for water corresponded to the actual water volume in the excised liver. The volume of distribution for the extracellular diffusible substances was shown to decrease with increase in molecular weight.     

Regional trapping of microspheres in the lung compares well with regional blood flow

Microspheres (MS) are often used to measure the distribution of pulmonary blood flow in the assumption that the number of MS trapped in a region is proportional to blood flow. However, regional distribution of trapped MS has not been directly compared with regional blood flow in the lung. Regional trapping of MS was compared with regional flow of erythrocytes (RBC's) in isolated, perfused left lungs of dogs. Radioactivity from labeled MS and RBC's was measured by external detection using a gamma camera. We defined six regions of interest in the image of the left lateral surface of the lung: a dorsocaudal, a caudal, two ventral, an apical, and a central region. In each lung, regional trapping of MS was measured from the image of radioactivity obtained after slow injection of a suspension of MS into the arterial perfusion tubing. A radioactive bolus of labeled RBC's was injected during rapid imaging of the lung to obtain radioactivity vs. time curves from each region. The peaks of the regional radioactivity vs. time curves were used to estimate regional flows, though compensation had to be made for overlap of the washout and washin phases of the bolus of labeled RBC's. The results indicated that there were no differences in the regional distribution of MS compared with the regional distribution of RBC flow in isolated, perfused dog lungs.     

Intratracheal gene transfer of tissue factor pathway inhibitor attenuates pulmonary fibrosis

Activation of the coagulation system and increased expression of tissue factor (TF) in pulmonary fibrosis associated with acute and chronic lung injury have been previously documented. In the present study, we evaluated the effect of TF inhibition with intratracheal gene transfer of tissue factor pathway inhibitor (TFPI), a potent and highly specific endogenous inhibitor of TF-dependent coagulation activation, in a rat model of bleomycin-induced lung fibrosis. Significant lung fibrotic changes as assessed by histologic findings and hydroxyproline content, and increased procoagulant activity and thrombin generation in bronchoalveolar lavage fluid were detected in rats after intratracheal injection of bleomycin. Intratracheal administration of an adenovirus vector expressing TFPI significantly decreased bleomycin-induced procoagulant and thrombin generation resulting in a strong inhibition of pulmonary fibrosis. TFPI-overexpression in the lung was associated with a significant reduction in gene expression of the connective tissue growth factor, a potent profibrotic growth factor. This is the first report showing that direct inhibition of TF-mediated coagulation activation abrogates bleomycin-induced pulmonary fibrosis.     

Suppression of norepinephrine appearance rate in plasma by diazepam in humans

Studies demonstrating benzodiazepine-induced reductions in plasma norepinephrine (NE) have assumed that changes in circulating plasma NE closely parallel changes in sympathetic nervous system (SNS) activity and that benzodiazepines suppress SNS outflow. However, decreases in plasma NE could also result from increased removal of NE from plasma via neuronal uptake or tissue metabolism. This study used a tritiated norepinephrine ([3H] NE) isotope dilution technique for measurement of plasma NE kinetics to determine if the fall in plasma NE induced by a single dose of diazepam orally administered to eight psychiatrically-healthy volunteers was due to a fall in plasma NE appearance rate or an increase in plasma NE removal. Diazepam decreased plasma NE appearance, but not clearance, and also decreased plasma epinephrine and mean arterial pressure, memory performance and alertness. Plasma levels of diazepam were correlated with drug effects on memory and alertness but not cardiovascular or SNS effects.     

A model for hypoxic constriction of the pulmonary circulation

The detailed anatomic and biodynamic data provided for the cat lung by Zhuang et al. (J. Appl. Physiol. 55: 1341-1348, 1983) allowed pressure-flow curves for the normal lung to be generated. This model has been modified to permit the stimulation of the pressure and flow distribution effects of hypoxic pulmonary vasoconstriction for a two-compartment lung and generalized to allow comparison with the experimental results from dogs (and probably other species). Hypoxic pulmonary vasoconstriction is simulated by reduction of the initial diameter of the smallest six orders of pulmonary arteries. Expressions are presented that relate the alveolar and mixed-venous O2 tensions to a graded constriction of these vessels. In addition, the diameter of the capillary sheet and the six small arteries is defined with a maximum diameter at a transmural pressure of 20 cmH2O. Pressure-flow curves are derived for any combination of alveolar and mixed-venous O2 tension, alveolar and pleural pressure, left atrial pressure, and hematocrit. The two-compartment model is solved by an iterative procedure to identify the distribution of the flow and the resulting pulmonary arterial pressure when the compartments differ by size, hypoxic constriction, or other imposed conditions. The results of the model are compared with those from a variety of experimental preparations. It is concluded that the model is useful for identifying the quantitative causes of changes in the response to hypoxic pulmonary vasoconstriction and for the exploration of the functional influence of mechanical properties of the vasculature.     

Differences in regional vascular conductances in isolated dog lungs

The distribution of pulmonary blood flow is influenced by gravity, regional lung expansion, and hypoxic pulmonary vasoconstriction. However, these factors cannot completely explain the three-dimensional distribution of blood flow in the lung. The present study was designed to see whether anatomically related factors could contribute. Regional blood pressure vs. flow curves were determined in 100-230 small parenchymal samples (0.3-0.4 ml) from 12 isolated perfused dog lungs held at constant inflation pressure. In each region four blood flows were measured using radioactively labeled microspheres, and the four corresponding regional perfusion pressures were determined by correcting the measured perfusion pressure for hydrostatic effects. There were considerable differences in the slopes of the pressure vs. flow curves among lung regions. Dorso-caudal regions of the lung had higher vascular conductances than ventrocephalad regions, independent of the vertical orientation of the lung or the inflation volume during injections of microspheres. Thus the distributions of regional vascular conductances were related to the anatomic location and were not related to gravity, nor were they caused by nonuniformities in regional lung expansion or by hypoxic vasoconstriction or edema.     

实验性肺纤维化大鼠发病过程中肺组织FIZZ1蛋白和mRNA表达的动态变化

为了探讨FIZZ1（found in inflammatory zone 1）在肺纤维化发病中的作用,应用博莱霉素（5mg／kg体重）气管内注入复制实聆性人鼠肺纤维化模犁,采用HE染色、Masson三联染色、羟脯氨酸含量测定、免疫组织化学染色、原位杂交等方法,观察实验性人鼠肺纤维化的发病过程及其肺组织中FIZZ1蛋白、mRNA表达水平的动态变化。结果显示：（1）实验性人鼠肺纤维化发病过程中,呈现舆型的肺泡炎（7d）、纤维组织增生（14～2ld）及稳定的肺纤维化（28d）等表现;（2）FIZZ1蛋白在正常肺组织表达较弱,存肺纤维化组7d时表达明显增强,14d时较7d时有所减弱,21及28d明显减弱;（3）FIZZ1 mRNA在正常肺组织巾表达较弱,在肺纤维化组7d时表达明显增强,14d时开始减弱,2l及28d明显减弱,但仍强于正常组。上述结果提示,FIZZ1蛋白和mRNA在实验性大鼠肺纤维化发病过程中呈现明显的动态变化,并可能参与了肺纤维化的发生。     

Expression of cardiac neural crest and heart genes isolated by modified differential display

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent outflow tract septation are critical events during vertebrate heart development. We have performed four modified differential display screens in the chick embryo to identify genes that may be involved in CNC, OFT, secondary heart field, and heart development. The screens included differential display of RNA isolated from three different axial segments containing premigratory cranial neural crest cells; of RNA from distal outflow tract, proximal outflow tract, and atrioventricular tissue of embryonic chick hearts; and of RNA isolated from left and right cranial tissues, including the early heart fields. These screens have resulted in the identification of the five cDNA clones presented here, which are expressed in the cardiac neural crest, outflow tract and developing heart in patterns that are unique in heart development.     

Pulmonary arterial transit times

To begin to characterize the pulmonary arterial transport function we rapidly injected a bolus containing a radiopaque dye and a fluorescence dye into the right atrium of anesthetized dogs. The concentrations of the dye indicators were measured in the main pulmonary artery (fluoroscopically) and in a subpleural pulmonary arteriole (by fluorescence microscopy). The resulting concentration vs. time curves were subjected to numerical deconvolution and moment analysis to determine how the bolus was dispersed as it traveled through the arteriole stream tube from the main pulmonary artery to the arteriole. The mean transit time and standard deviation of the transport function from the main pulmonary artery to the arterioles studied averaged 1.94 and 1.23 s, respectively, and the relative dispersion (ratio of standard deviation to mean transit time) was approximately 64%. This relative dispersion is at least as large as those reported for the whole dog lung, indicating that relative to their respective mean transit times the dispersion upstream from the arterioles is comparable to that taking place in capillaries and/or veins. The standard deviations of the transport functions were proportional to their mean transit times. Thus the relative dispersion from the main pulmonary artery to the various arterioles studied was fairly consistent. However, there were variations in mean transit time even between closely adjacent arterioles, suggesting that variations in mean transit times between arteriole stream tubes also contribute to the dispersion in the pulmonary arterial tree.     

Correlation of pulmonary ACE activity and capillary surface area during postnatal development

Although a considerable amount of information is available regarding the remodeling and growth of the pulmonary arterial circulation, relatively little is known regarding postnatal development of the pulmonary microcirculation. We hypothesized that the maximal velocity (Vmax) of pulmonary angiotensin-converting enzyme (ACE) activity, measured from indicator-dilution outflow curves using a synthetic substrate, 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), is directly related to the capillary endothelial cell surface area in the lungs of developing lambs. Accordingly we measured apparent kinetics of pulmonary ACE activity in 22 anesthetized ventilated lambs (2-171 days old) and compared our functional assessment to simultaneous in vivo determinations of CO diffusing capacity (DLCO) and postmortem structural assessment of alveolar septal dimensions using stereology and electron microscopy. There was a progressive increase in Vmax of ACE in this age group, with little change in apparent affinity for BPAP. Similar functional manifestation of growth was noted by an age-dependent increase in DLCO. Neither Vmax nor DLCO was significantly affected by an increase in left atrial pressure to 19 Torr (via inflation of a balloon in the left atrium), suggesting little recruitment of vessels under conditions of the present protocol. A close correlation was observed when either Vmax for ACE activity or DLCO was plotted vs. capillary endothelial cell surface area. Double logarithmic transformation of capillary endothelial cell surface area, Vmax-ACE and DLCO vs. lung volume revealed power functions with slopes all greater than that predicted from isotropic growth, suggesting selective differential postnatal development of the endothelium of the alveolar septum in lambs from 2-171 days of age.