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We have previously demonstrated that the detergent Tyloxapol is effective in preventing reactions to endotoxin. We studied the effects of Tyloxapol on the morbidity and mortality from endotoxemia in rabbits and on the mortality in rats with sepsis. The effects of Tyloxapol on endotoxin binding and macrophage activation were studied in the macrophage cell line RAW264.7 and CHO cells expressing CD14. Isolated human leukocytes were used to study the effects of Tyloxapol on immune reactions, leukocyte motility, and phagocytosis. Intravenous Tyloxapol (200 mg/kg), given prior to or at the time of endotoxin infusion protected rabbits from developing shock. In rats with peritoneal sepsis, a lipid-rich diet and Tyloxapol given at the time of induction of peritonitis protected them from septic death. In vitro, Tyloxapol blocked the binding of endotoxin to murine macrophages and CHO cells expressing CD14, activation of macrophages, and also some antigen-antibody immune reactions (mediated by CD2, CD4, CD22, HLA-DR). Tyloxapol may prevent the reaction to endotoxin by desensitizing endotoxin-recognizing receptors.

Sheep have reactive pulmonary intravascular macrophages, which are essential for the marked pulmonary vascular response to infusions of small quantities of endotoxin. In another species with reactive pulmonary intravascular macrophages, horses, our laboratory found that an intravenous biosafe detergent, tyloxapol, inhibited some systemic and pulmonary responses to endotoxin (Longworth KE, Smith BL, Staub NC, Steffey EP, and Serikov V. Am J Vet Res 57: 1063-1066, 1996). We determined whether the same detergent would inhibit endotoxin responses in awake sheep. In 10 awake, instrumented sheep with chronic lung lymph fistulas, we did a control experiment by intravenously infusing 1 microg/kg Escherichia coli endotoxin. One week later, we gave 40 micromol/kg tyloxapol intravenously 1-4 h before infusing the same dose of endotoxin. In these paired studies, we compared pulmonary hemodynamics, lung lymph dynamics, body temperature, circulating leukocyte concentrations, and circulating tumor necrosis factor for 6 h. In all 10 sheep, tyloxapol blocked 80-90% of the pulmonary responses and 70-90% of the systemic responses. Tyloxapol is safe, inexpensive, easy to use, and effective immediately. It may be a clinically useful approach to contravening many of the effects of endotoxemia, in humans as well as animals.

We recently showed that we can selectively and safely deplete most (average 85%) of the pulmonary intravascular macrophages in sheep by intravenously infusing liposomes containing dichloromethylene bisphosphonate. After a 1-h stable baseline, we made a 6-h comparison after a 30-min intravenous endotoxin infusion (1 microg/kg) between six anesthetized control lambs and six anesthetized lambs in which the intravascular macrophages had been depleted 24 h previously. Three of the control lambs had been macrophage depleted and allowed to recover their intravascular macrophage population for >/=2 wk. After depletion, both the early and late pulmonary arterial pressure rises were dramatically attenuated. Our main interest, however, was in the acute lung microvascular injury response. The early and late rises in lung lymph flow and the increase in lung lymph protein clearance (lymph flow x lymph-to-plasma protein concentration ratio) were >90% attenuated. We conclude the pulmonary intravascular macrophages are responsible for most of the endotoxin-induced pulmonary hypertension and increased lung microvascular leakiness in sheep, although the unavoidable injury of other intravascular macrophages by the depletion regime may also contribute something.

We previously showed in newborn lambs that the pulmonary hemodynamic responses to foreign particulate matter (liposomes; Monastral blue) developed in parallel with the maturation of the pulmonary intravascular macrophage system. We now report our use of the liposome-encapsulated heavy-metal-chelating agent dichloromethylene diphosphonate to deplete the intravascular macrophages of small lambs. Functionally and by quantitative histology, we depleted the vast majority of the intravascular macrophages (71% by Monastral blue particle retention, n = 22; 77% by histology; n = 2). Depletion success increased to > 90% as we optimized the liposome-depletion regime. Recovery of the lung hemodynamic response began within 3 days. By 2 wk, the functional responses had fully recovered (n = 8), and, according to quantitative histology, the macrophage population (n = 2) had recovered 65%. Macrophage depletion in lambs is relatively inexpensive and easy to achieve. It is a safe procedure and is followed by full recovery in approximately 2 wk, provided that an aseptic technique is used to prevent bacteremia.

The objective of this study was to investigate the influence of volume ventilation (VE) and cardiac output (Q) on the temperature of the expired gas at the distal end of the endotracheal tube in anesthetized humans. In 63 mechanically ventilated adults, we used a step decrease in the humidity of inspired gas to cool the lungs. After change from humid to dry gas ventilation, the temperature of the expired gas decreased. We evaluated the relationship between the inverse monoexponential time constant of the temperature fall (1/tau) and either VE or Q. When VE was increased from 5.67 +/- 1.28 to 7.14 +/- 1.60 (SD) l/min (P = 0. 02), 1/tau did not change significantly [from 1.25 +/- 0.38 to 1.21 +/- 0.51 min-1, P = 0.81]. In the 11 patients in whom Q changed during the study period (from 5.07 +/- 1.81 to 7.38 +/- 2.45 l/min, P = 0.02), 1/tau increased correspondingly from 0.89 +/- 0.22 to 1. 52 +/- 0.44 min-1 (P = 0.003). We calculated the airway thermal volume (ATV) as the ratio of the measured values Q to 1/tau and related it to the body height (BH): ATV (liters) = 0.086 BH (cm) - 9. 55 (r = 0.90).

Background: Pulmonary intravascular macrophages are resident cells in the pulmonary circulation of sheep. Sheep, unlike species without pulmonary intravascular macrophages, exhibit pulmonary hypertension in response to intravenously injected particles. We reported in lambs that pulmonary vascular reactivity to intravenous particles increases with age as the population of intravascular macrophages develops. Preliminary quantitative histologic data showed that newborn lambs are born with few intravascular macrophages, but a large population develops over 2 weeks after birth. In this study, we present a complete quantitative analysis at the ultrastructural level., Methods: We fixed five newborn and five 2-week-old lamb lungs by vascular perfusion and examined the tissue by electron microscopy., Results: The fraction of capillary lumen taken up by intravascular macrophages/ monocytes is about three times greater in the lungs of 2-week-old lambs than that in newborn lambs (16% vs. 5%; P < 0.05). The fraction of capillary surface density associated with intravascular macrophages/monocytes is about three times greater in 2-week-old lambs than that in newborn lambs (8% vs. 3%; P < 0.05). The number of macrophages more than doubles with age (16 +/- 4 vs. 7 +/- 2; P < 0.05) and the estimated size (volume-weighted mean volume) increases by more than 1.5 times (294 +/- 46 microns 3 vs. 184 +/- 29 microns 3; P < 0.05)., Conclusions: These data agree closely with Monastral blue retention by the lung (reported previously); there are more than twice as many mature pulmonary intravascular macrophages at 2 weeks than at 1 day after birth, and the cells are 1.5 times larger.

Interleukin-2 (IL-2) is reputed to cause a "vascular leak syndrome." We studied pulmonary hemodynamics and lymph dynamics in six sheep treated for 7 days with IL-2 (1.8 million IU/kg twice daily or 1.8 million IU/kg each day as a continuous infusion). Lung lymph flow increased from 4.8 +/- 2 ml/15 min pre-IL-2 to 14.4 +/- 6.8 ml/15 min on the seventh day of IL-2. The lymph-to-plasma protein concentration ratio was unchanged (0.70 +/- 0.06 vs. 0.63 +/- 0.13). The plasma-to-lymph equilibration half-time of radiolabeled albumin was 2.0 +/- 0.6 h pre-IL-2 and 1.0 +/- 0.7 h on day 7 of IL-2. Pulmonary arterial pressure was 24 +/- 7 cmH2O pre-IL-2, increased to 32 +/- 4 cmH2O on the fourth day of IL-2, and returned to 29 +/- 5 cmH2O on the seventh day of IL-2. Extravascular lung water was normal (4.07 +/- 0.25 g/g dry lung). To clearly determine whether the increase in lung lymph flow was due to hemodynamic changes or to increased leakiness of the microvascular barrier, we volume loaded six sheep with lactated Ringer solution before and after 3 days of IL-2 treatment (1.8 million IU/kg twice daily). Lung lymph flows increased fivefold during 4 h of crystalloid infusion compared with baseline and were higher after 3 days of IL-2. However, lymph-to-plasma protein concentration ratios decreased to the same low levels pre-and post IL-2 (0.39 +/- 0.06 vs. 0.41 +/- 0.10), indicating and intact microvascular barrier. Extravascular lung water was elevated (5.56 +/- 0.39 g/g dry lung) but was not different from lung water in three volume-loaded control sheep (4.87 +/- 0.53 g/G dry lung). We conclude that IL-2 causes minimal or no injury to the pulmonary microvascular barrier and that volume expansion during IL-2 treatment can cause hydrostatic pulmonary edema.

Objective: To determine whether a detergent can prevent most of the early effects of i.v. infusion with Escherichia coli endotoxin (< 100 ng/kg of body weight) in horses: marked pulmonary hypertension, acute leukopenia, and fever., Animals: 8 healthy adult horses (4 male, 4 female), 415 to 615 kg., Design and Procedure: Control and detergent experiments were performed in each horse while it was awake but sedated. In control experiments, 10 to 100 ng of E coli endotoxin/kg was given. In detergent experiments, 100 mg of detergent/kg was given 1 hour before injecting endotoxin, similar to the control experiments., Results: In control experiments, pulmonary arterial pressure increased transiently over 40 minutes by 33 +/- 8 mm of Hg (mean +/- SD; P < 0.001), then returned to baseline. Circulating leukocytes decreased to 47 +/- 19% (P < 0.02) of baseline by 1 hour after endotoxin, then increased above baseline by 6 hours. Rectal temperature increased by 0.7 +/- 0.4 C (P < 0.01). In detergent experiments, the increase in pulmonary arterial pressure was much less than that in the control experiments (8 +/- 7 mm of Hg; P < 0.001). Circulating leukocytes did not decrease, and the increase in rectal temperature after endotoxic was blocked., Conclusions: This attenuation of te response to endotoxin may occur because the normal steps in the response of pulmonary intravascular macrophages (ie, endocytosis of endotoxin and subsequent release of inflammatory mediators) are altered by the detergent. This low-technology, inexpensive, and safe treatment could be an important new clinical tool for veterinarians in combating endotoxemia.

Seven horses (4 anesthetized and 3 awake) and 2 ponies (anesthetized) were studied to evaluate the high sensitivity of the pulmonary circulation of the horse to various blood-borne particles, and to establish the presence of intravascular macrophages in the lung. Pulmonary and systemic pressures and cardiac output before and during particle injection were measured in some animals. An anesthetized foal had a large increase in pulmonary arterial pressure (32 and 34 mm of Hg) within 1 minute of IV administration of small test doses of radioactively labeled liposomes (2.5 mumol/kg of body weight) or a 1% suspension of blue pigment (0.3 ml/kg), respectively. Quantitative real-time gamma camera imaging of the foal revealed high retention of the labeled liposomes during the first pass through the lungs; retention persisted throughout the experiment. Postmortem analysis revealed 55 and 47% lung retention of liposomes and blue pigment, respectively. The 2 anesthetized ponies had increased pulmonary artery pressure of 34 +/- 7 mm of Hg, decreased cardiac output, and 42% lung retention after administration of 1% blue pigment (0.2 ml/kg), whereas 3 awake horses had increased pressure of 28 +/- 9 mm of Hg after 1.8 x 10(8) (1.8-microns-diameter) latex microspheres/kg. None of the injected particles caused vascular obstruction, and they do not cause pulmonary vascular reactivity in species that lack pulmonary intravascular macrophages. Finally, 3 horses (1 anesthetized and 2 awake) were infused IV with small doses of the blue pigment, and their lungs were perfusion-fixed to identify specific labeling of the pulmonary intravascular macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 (IL-2) is reputed to cause pulmonary microvascular injury. We studied the pulmonary and splanchnic microcirculation of anesthetized sheep after one dose (1.8 x 10(6) IU/kg) of IL-2 (n = 9) and after six doses (1.8 x 10(6) IU.kg-1.dose-1) of IL-2 over 3 days (n = 9). Seven control sheep received only 5% dextrose diluent. We measured hemodynamics and lymph dynamics in anesthetized sheep after the final dose of IL-2 or diluent. After one dose of IL-2, caudal mediastinal node (mainly pulmonary) lymph flow was stable, whereas thoracic duct lymph flow increased from a baseline of 54 +/- 6 to 124 +/- 22 ml/h. After 3 days of IL-2, the caudal mediastinal node lymph flow increased from 7.7 +/- 5.5 to 19.0 +/- 14.8 ml/h 5-6 h after the final dose of IL-2, and thoracic duct lymph flow increased from 84 +/- 43 to 143 +/- 42 ml/h. The lymph-to-plasma protein concentration ratio increased after IL-2 for thoracic duct but not for caudal mediastinal node lymph. The equilibration rate of 125I-albumin from plasma to caudal mediastinal node lymph did not change, whereas plasma-to-thoracic duct lymph equilibration was faster after both one dose and 3 days of IL-2. Positron emission tomography showed no increase in the pulmonary transcapillary escape rate for 68Ga-labeled transferrin or in extravascular lung water (n = 4). We conclude that IL-2 at doses two to three times those used clinically does not significantly injure the pulmonary microcirculation of sheep.

Transfusion of small quantities of heterologous blood may cause severe pulmonary hypertensive response in certain species. To determine the responsible component in the donor blood and the main mediator, we studied the responses of goats to small quantities of rabbit blood components and observed the effects of several pharmacologic agents on these responses. In anesthetized goats, a bolus injection of 0.004 ml/kg rabbit blood caused the pulmonary arterial pressure to increase from 25.3 +/- 2.8 to 57.1 +/- 11.6 cm H2O within 45 to 90 s, and the aortic thromboxane concentration rose from 44 +/- 38 to 238 +/- 104 pg/ml. Pulmonary vascular resistance increased more than 4-fold, whereas systemic vascular resistance increased moderately (50%). The erythrocyte stroma, mainly cell membranes, caused similar responses; other blood components were all ineffective. By blocking the production of thromboxane, indomethacin and U63557A (thromboxane synthetase inhibitor) abolished nearly all of the hemodynamic responses to rabbit blood. Isoproterenol also largely attenuated the responses to rabbit blood by blocking thromboxane production without interfering with the responses to the thromboxane mimic U46619. Nitrendipine (calcium-channel blocker) equally attenuated rabbit blood and U46619-induced hemodynamic responses but did not block thromboxane production. Chlorpheniramine (H1-receptor antagonist) partially blocked the hemodynamic responses to rabbit blood without affecting thromboxane production or U46619-induced responses. We conclude that the erythrocyte membrane is the responsible component in the donor blood and thromboxane is the predominant mediator. The main action of isoproterenol is to reduce thromboxane production and histamine participates by possible interaction with cyclooxygenase products.

We sought to determine whether pulmonary intravascular macrophages are involved in pulmonary vascular sensitivity to intravenously injected particles in sheep. We estimated that newborn lambs have few of these macrophages at birth but develop a 10-fold greater density within 2 wk. Awake, chronically instrumented newborn lambs showed no change in pulmonary vascular driving pressure (pulmonary arterial minus left atrial pressure) after injection of either liposomes [2 +/- 3 (SD) cmH2O; n = 5] or Monastral blue particles (3 +/- 2 cmH2O; n = 6) and showed no net pulmonary production of thromboxane B2, the stable metabolite of the vasoconstrictor thromboxane A2. In contrast, five of those lambs 2 wk later showed both an increase in pulmonary vascular driving pressure after injection of liposomes and Monastral blue (20 +/- 16 and 25 +/- 15 cmH2O, respectively; P < 0.05) and net pulmonary production of thromboxane B2 (171 +/- 103 and 429 +/- 419 pg/ml plasma, respectively; P < 0.05). Older lambs (n = 5) had higher pulmonary uptakes than newborn lambs (n = 6) of radioactive liposomes (47 +/- 13 vs. 12 +/- 10%; P < 0.01) and Monastral blue (53 +/- 6 vs. 21 +/- 10%; P < 0.05). We conclude that pulmonary intravascular macrophages are responsible for the sensitivity of sheep to intravenous foreign particles and are essential for a cascade of processes leading to microvascular injury.

We have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent.

We tested the accuracy, sensitivity, and reproducibility of a new lung water computer, based on the thermal conductivity technique, in 22 anesthetized closed-chest ventilated sheep with different treatments: 1) controls (n = 8), 2) 0.05 ml/kg of oleic acid + 100 ml/kg of lactated Ringer solution (n = 6), and 3) airway instillation of saline [3.1 +/- 1.3 (SD) g/kg, n = 8]. After 4 h, we determined the extravascular lung water gravimetrically. We found a significant overall correlation between the final extravascular lung thermal volume and the gravimetric extravascular lung mass (P < 0.001). Although the average ratio of extravascular lung thermal volume to extravascular lung mass was 0.97 +/- 0.25 ml/g for all groups, the computer overestimated extravascular lung mass in controls by 10% (17 g) and underestimated it in sheep with oleic acid by 15% (95 g) and in sheep with airway instillation by 8% (37 g). The computer also underestimated the small quantities of saline placed via the airway in the alveolar space by 75% (61 g). Reproducibility of three consecutive measurements was 4.3% (SE). We conclude that the thermal conductivity technique has an ability to detect the baseline extravascular lung mass but has a poor ability to detect an accurate increment of the extravascular lung water under poor tissue perfusion in anesthetized ventilated sheep.

We tested the effect of interstitial edema on lung lymph flow when no filtration occurred. In 16 anesthetized open-thorax ventilated supine goats, we set pulmonary arterial and left atrial pressures to nearly zero and measured lymph flow for 3 h from six lungs without edema and ten with edema. Lymph flow decreased exponentially in all experiments as soon as filtration ceased. In the normal lungs the mean half time of the lymph flow decrease was 12.7 +/- 4.8 (SD) min, which was significantly shorter (P less than 0.05) than the 29.1 +/- 14.8 min half time in the edematous lungs. When ventilation was stopped, lymph flow in the edematous lungs decreased as rapidly as in the normal lungs. The total quantity of lymph after filtration ceased was 2.7 +/- 0.8 ml in normal lungs and 9.5 +/- 6.3 ml in edematous lungs, even though extravascular lung water was doubled in the latter (8.4 +/- 2.4 vs. 3.3 +/- 0.4 g/g dry lung, P less than 0.01). Thus the maximum possible clearance of the interstitial edema liquid by the lymphatics was 6.3 +/- 4.8%. When we restarted pulmonary blood flow after 1-2 h in four additional goats, lymph flow recovered within 30 min to the baseline level. These findings support the hypothesis that lung lymph flow originates mainly from alveolar wall perimicrovascular interstitial liquid and that the contribution of the lung lymphatic system to the clearance of interstitial edema (bronchovascular cuffs, interlobular septa) is small.

A noninvasive method for measuring pulmonary blood flow and lung mass (called airway thermal volume), based on the measurements of lung heat exchange with environment, is described. The lungs function as a steady-state heat exchange system, having an inner heat source (pulmonary blood flow) and an external heat sink (ventilation). Sudden changes in the steady-state condition, such as caused by hyperventilation of dry air, lead to a new steady state after a few minutes. The expired air temperature difference between the initial and final steady states is proportional to pulmonary blood flow, whereas the rate at which the new steady state is achieved is proportional to airway thermal volume. The method was tested in 20 isolated dogs lungs, 9 perfused goat lungs, and 27 anesthetized sheep. The expired air temperature fall during hyperventilation was inversely proportional to the perfusion rate of the isolated lungs, and half-time of the temperature fall was proportional to the lung tissue mass. Experiments in anesthetized sheep showed that the measured airway thermal volume is close to the total mass of the excised lungs, including its residual blood (r = 0.98). Pulmonary edema and fluid instillation into the bronchial tree increased in the measured lung mass.

We have found that the goat is extraordinarily sensitive to very small quantities of rabbit or rat blood. As little as 0.004 ml/kg induces transient pulmonary hypertension [maximal rise in pulmonary arterial pressure 32 +/- 10 (SD) cmH2O] in goats. We hypothesized that this reaction may be related to the presence of the resident population of intravascular macrophages that reside in the pulmonary capillaries of goats. If that is so, then rabbits or rats, which have few or no intravascular macrophages, should not be reactive to foreign blood. We compared pulmonary hemodynamics and changes in blood thromboxane B2 concentrations among goats, rabbits, and rats in response to graded doses of foreign blood. The pulmonary reaction to foreign blood was much greater in goats than in rabbits or rats, even though we injected up to 10- or 60-fold larger amounts into the latter species. In goats the pulmonary vascular pressure response to rabbit blood was dose dependent in goats and correlated well with changes in systemic arterial thromboxane B2 concentrations [change in pulmonary arterial pressure = 0.07 (thromboxane B2) + 8.3, r = 0.79]. We also tested the prostaglandin H2 endoperoxide analogue (U-46619) and found that the goats are somewhat more reactive than rabbits. We conclude that the pulmonary hemodynamic reaction to foreign blood is consistent with the concept that the foreign erythrocytes are reacting with the pulmonary intravascular macrophages in goats. The lower reactivity of the rabbit pulmonary circulation to thromboxane may also have a role.

Controversy continues about the contribution of the veins to pulmonary vascular resistance. From data obtained in studies using intravascular catheters, it appears that a major fraction (up to 44%) of the total pulmonary vascular pressure drop resides in larger (greater than 1.0 mm diam) veins, whereas micropuncture data and various models give much less pressure drop. Theoretically, artifactual pressure drops can be obtained if an intravascular catheter partly obstructs the vessel. We made measurements of pressure in the same lung vein with two different-sized catheters (1.2 and 0.6 mm OD, respectively). In paired experiments the larger catheter always measured a higher pressure than the smaller one, except close to the large lobar vein outlet. In some of the experiments we measured the diameter of the vessel containing the indwelling catheter by freezing the lung and then serial-sectioned the frozen lung. From these data we could infer that the range of vein diameter in the which the smaller catheter measured a lower pressure was 1.5-4 mm. We conclude that the larger catheter overestimated the pressure because of greater obstruction. The pressures obtained with the smaller catheter suggest that little (less than 10%) of the total pulmonary vascular resistance resides in veins larger than approximately 1 mm diam under zone 3 baseline conditions.

The equations developed by Landis and Pappenheimer (Handbook of Physiology. Circulation, 1963, p. 961-1034) for calculating the protein osmotic pressure of human plasma proteins have been frequently used for other animal species without regard to the fractional albumin concentration or correction for protein-protein interaction. Using an electronic osmometer, we remeasured the protein osmotic pressure of purified sheep albumin and sheep plasma partially depleted of albumin. We measured protein osmotic pressures of serial dilutions over the concentration range 0-180 g/l for albumin and 0-100 g/l for the albumin-depleted proteins at room temperature (26 degrees C). Using a nonlinear least squares parameter-fitting computer program, we obtained the equation of best fit for purified albumin, and then we used that equation together with the measured albumin fraction to obtain the best-fit equation for the nonalbumin proteins. The equation for albumin is IIcmH2O,39 degrees C = 0.382C + 0.0028C2 + 0.000013C3, where C is albumin concentration in g/l. The equation for the nonalbumin fraction is IIcmH2O,39 degrees C = 0.119C + 0.0016C2. Up to 200- and 100-g/l protein concentration, respectively, these equations give the least standard error of the estimate for each of the virial coefficients. The computed number-average molecular weight for the nonalbumin proteins is 222,000. Using the new equations, we constructed a nomogram, based on the one of Nitta and co-workers (Tohoku J. Exp. Med. 135: 43-49, 1981). We tested the nomogram using 144 random samples of sheep plasma and lymph from 31 sheep. We obtained a correlation coefficient of 0.99 between the measured and nomogram estimates of protein osmotic pressure.

Pleural space width was measured by four morphological approaches using either frozen hydrated or freeze-substituted blocks of chest wall and lung. Anesthetized sheep were held in the lateral (n = 2), sternal recumbent (n = 2), or vertical (head-up; n = 2) position for 30 min. The ribs and intercostal muscles were excised along a 20-cm vertical distance of the chest wall region, which was sprayed with liquid Freon 22, cooled with liquid nitrogen, to facilitate the fastest possible freezing of the visceral and parietal pleura. We measured pleural space width in frozen hydrated blocks by reflected-light and low-temperature scanning electron microscopy and in freeze-substituted, fixed, and embedded tissue blocks by light and transmission electron microscopy. We combined the data from the two groups of sheep held sternally recumbent and vertical because the results were comparable. The average arithmetic mean data for pleural space width determined by reflected-light analysis for samples near the top (18.5 microns) and bottom (20.3 microns) of the chest, separated by 15 cm of lung height, varied inversely with lung height (n = 4; P less than 0.009). The average harmonic mean data demonstrated a similar gravity-dependent gradient (17.3 and 18.8 microns, respectively; P less than 0.02). Therefore a slight vertical gradient of approximately -0.10 micron/cm of lung height was found for costal pleural space width. Pleural space width in the most dependent recesses, such as the costodiaphragmatic recess, reached 1-2 mm. We never found any contacts between the visceral and parietal pleura with either of the frozen hydrated preparations. No points of mesothelial cell contact were revealed in the light- and transmission electron microscopic views of the freeze-substituted tissue, despite an apparent narrower pleural space associated with the tissue-processing steps. We conclude that the pleural space has a slightly nonuniform width, contacts if they occur must be very infrequent, and pleural liquid clearance is probably facilitated by liquid accumulation in dependent regions where lymphatic pathways exist.

We studied the effect of intravenous dextran 70 infusion on lung liquid and protein exchange to determine whether its effects were due to altered hemodynamics or to altered microvascular permeability. In each of six instrumented awake sheep with chronic lung lymph fistulas, we performed three experiments: 1) control, 2) a 30-minute infusion of 1 l of 6% dextran 70, and 3) an infusion of 1 l of 0.9% NaCl. In addition to pulmonary hemodynamics and lymph dynamics, we measured the plasma-to-lung lymph equilibration rate of [125I] albumin. We followed all the sheep for 10 hours, including a 2-hour baseline period. Dextran was more effective in expanding plasma volume (63 +/- 15% [mean +/- SD]) than saline (11 +/- 6%) at the end of the 30-minute infusion. Pulmonary vascular pressures increased after dextran and remained elevated for 8 hours, whereas after saline the pressures returned to baseline within 1 hour. After dextran, lung lymph flow increased and remained elevated. It was only transiently increased after saline. We confirmed that dextran equilibrated rapidly with lung lymph (half-time, less than 0.6 hour), even though it maintained plasma volume expansion for the whole body (half-time, 11.1 +/- 2.7 hours). The dextran increased both plasma and lymph total macromolecular osmotic pressure but did not increase the plasma-interstitial (lymph) osmotic pressure difference in the lung, except transiently during the infusion. The lymph/plasma protein concentration ratio increased after dextran due mainly to plasma protein dilution. There were no differences in the half-time of tracer albumin equilibration between plasma and lung lymph (control, 2.2 +/- 0.6 hours; saline, 2.0 +/- 0.6 hours; dextran, 2.3 +/- 0.6 hours). Dextran 70 increased liquid filtration mainly by increasing microvascular pressure and possibly filtration surface area. There was no evidence for a change in the leakiness of the lung microvascular barrier to albumin.

In paired experiments, we studied the effects of high-dose methylprednisolone on the acute pulmonary injury caused by 4 h of venous air embolization in 19 chronically instrumented, unanesthetized sheep with lung lymph fistulas. We compared the effect of methylprednisolone (30 mg/kg intravenous bolus) given before embolization, early (1 H) in the course of embolization, late (3 h) in the course of embolization, or after embolization (at the beginning of the recovery period). We measured pulmonary hemodynamics and lymph dynamics. In six sheep we also fixed lung tissue for semiquantitative histology, and in some we measured leukocyte concentrations in blood and in pulmonary lymph. Methylprednisolone did not significantly affect pulmonary hemodynamics but it largely prevented lung injury when it was given before embolization. It also lessened the degree of lung injury when it was given during embolization, although this effect became less marked as treatment was delayed. Methylprednisolone had no effect on lung injury when given after embolization was completed (4 h). We found fewer leukocytes attached to air emboli and fewer endothelial cell gaps in the lungs of sheep given methylprednisolone as prophylaxis. Leukocyte counts were lower in lung lymph and higher in the circulating blood of methylprednisolone-treated sheep. We conclude that methylprednisolone has a preventive effect on air embolism lung injury, such that its effect is greater when given earlier during the development of injury.

To determine whether lung edema leaks into the pleural space, we measured flow rates of visceral pleural liquid from exposed sheep lungs during volume loading and then compared the protein concentration of visceral pleural liquid and lung interstitial liquids (lymph and peribronchovascular cuff liquid). For 4 h, we volume loaded 24 anesthetized ventilated sheep with one side, both sides, or neither side of the chest open. During the experiment, we collected visceral pleural liquid from a bag surrounding the exposed lung and lung lymph; after the experiment, we collected peribronchovascular cuff liquid. We found that during volume loading visceral pleural liquid flow increased significantly by 2 h, and its protein concentration over the final hour was the same as that of lung interstitial liquids. The volume of visceral pleural liquid correlated with excess lung water and wedge pressure elevation. By our estimates, clearance of edema from the lung into the pleural space constituted 23-29% of all edema liquid collected, similar to measured lymph edema clearance. We conclude that edema liquid leaks directly from edematous sheep lungs into the pleural space and that this leakage provides an important additional route of edema clearance.

Until now, direct micropuncture measurements of vascular pressure in lung have been limited to small vessels less than 100 microns on the pleural surface. On the other hand, direct pressure measurements using small catheters (less than 1-mm OD) in pulmonary vessels have been limited to those greater than 1.2 mm. We measured pressure in intermediate-sized microvessels (300-700 microns) using the micropuncture method in isolated perfused rabbit lungs. These microvessels are located 2 or 3 mm beneath the pleura. We exposed them by microsurgery and punctured the relatively thick-walled vessels with specially configured micropipettes. We exposed one pulmonary microvessel in each rabbit lung by microsurgery on the left middle lobe. In 15 rabbit lungs we measured pressure in a total of six small arteries (275- to 470-microns diam) and nine small veins (300- to 700-microns diam) under high zone 3 conditions, near the zone 2/3 boundary. We found approximately 35% of the total pulmonary vascular pressure drop in arteries greater than 275-microns diam and 7% in veins greater than 300-microns diam. In veins greater than 500-microns diam, there was no measurable pressure drop. After the measurements, we froze the lung and confirmed that there was no detectable interstitial or alveolar edema in the cross sections of the punctured site. Our data are compatible with those of other investigators who have used isolated perfused rabbit lungs under similar experimental conditions.

The filtration variables, K (filtration coefficient), Ppmv (perimicrovascular pressure) and sigma (reflection coefficient), were estimated independently in previous reports using the Starling equation or the micropuncture method. We used matrix algebra to estimate these variables simultaneously. We measured filtration rate (Q) by a gravimetric method in isolated rat lung lobes in zone 1 conditions (alveolar pressure = 20 cmH2O) at two vascular pressures, Pvasc = 15 or 18 cm H2O and perfused the lobes with plasma containing a low or a high concentration of protein. By extrapolating the log of the rate of weight gain to t = 0, we obtain the initial filtration rate before any of the pressure variables (microvascular and perimicrovascular hydrostatic pressures) in the Starling equation changed. Assuming that protein filtered into perimicrovascular space only by convection, we substituted it into the Starling equation as follows: Q = K [(Pmv -- Ppmv) -- sigma 2 (IImv)], where Pmv and IImv are microvascular and perimicrovascular plasma protein osmotic pressures. IImv was estimated by Yamada's equation (Yamada et al. 1985). For the matrix algebra, we used three values, we omitted the value for the high protein, low vascular pressure experiment. We obtained K = 26.3 [mg/(min x cmH2O x g wet weight)], Ppmv = 6.2 cmH2O and sigma = 0.46. These values agree with values from previous reports. Since these 3 filtration variables are interrelated, this new method for simultaneous measurement is more accurate than independent measurements are. The chief advantage of this method is that it does not require a separate estimate of isogravimetric pressure or a direct measurement of interstitial pressure, and all variables are obtained simultaneously.

To determine whether intrathoracic nonpulmonary structures (caudal mediastinal node or esophagus) are reputed to affect lung lymph dynamics, we studied anesthetized, open-thorax, prone, ventilated sheep in which all lung afferent lymphatics to the caudal mediastinal node were eliminated. When we increased left atrial pressure by 20 cmH2O in four sheep, there was no effect on caudal mediastinal node efferent lymph flow or protein concentration, thus providing the completeness of the surgical preparation. In four sheep, intravenous histamine infusions (3 micrograms base.kg-1.min-1) had no effect on caudal mediastinal node lymph flow or protein concentration. In seven sheep with intact lung lymphatics, Escherichia coli endotoxin infusion (1 microgram/kg over 20 min) increased lymph flow with high lymph protein concentration during the late phase (2-6 h). In seven sheep, after all lung lymphatic afferents had been cut, endotoxin did not affect caudal mediastinal node lymph flow, although lymph protein concentration was decreased in the early 0-2 h, "hypertensive") phase. We conclude that at the concentrations tested, which are those regularly used in sheep lung experiments, the effects of histamine and endotoxin on caudal mediastinal node lymph flow and protein concentration are limited to the lung.

Five mongrel dogs (2 interstitial and 3 alveolar edema) were studied. Lung mechanics were measured by recording the flow, volume, and esophageal pressure according to the standard technique. Edema was produced by infusion of Ringer lactate solution. Lung sounds were recorded on tape from the dependent part of the chest wall. Lung sound signals were high-pass filtered at 100 Hz and subjected to fast Fourier transform. Samples of lung sounds were analyzed before (control) and at 5, 10, 20, 30, and 40 min after the infusion. The mean, median, and mode frequencies of sound power spectra at the control time were, respectively, 169.6 +/- 29.19, 129.6 +/- 29.81, and 136.0 +/- 29.87 (SD) Hz. These values increased significantly at 5 min after infusion to 194.0 +/- 26.08 (P less than 0.0037), 150.2 +/- 23.48 (P less than 0.0085), and 164.6 +/- 28.74 Hz (P less than 0.02), respectively. These values stayed significantly elevated at 10, 20, 30, and 40 min. The pulmonary wedge pressure, lung dynamic compliance, and pulmonary resistance were measured also at the same times. The mean, median, and mode frequencies correlated with pulmonary wedge pressure (P less than 0.00001, P less than 0.0001, P less than 0.0001), lung dynamic compliance (P less than 0.001, P less than 0.0001, P less than 0.0001), and pulmonary resistance (P less than 0.00001, P less than 0.00001, P less than 0.0001), respectively. There were no significant adventitious sounds up to 40 and 50 min after infusion. We concluded that pulmonary congestion and early edema alter the frequency characteristics of lung sounds early, before the occurrence of adventitious sounds. These altered lung sounds may be used as an index of pulmonary congestion and impending edema.

Although research on microcirculation has expanded rapidly in the last 20 years, clinical evaluation of microcirculation, as an entity, has not achieved any status in the United States. Three aspects of microcirculation are presented, wherein clinical applications exist or on the verge of existing. Biomicroscopy is the direct observation of, and measurements within, the microcirculation is superficial areas of the body. Not only can the pattern of the microcirculation be examined and related to microcirculatory function but also the intravascular relationships among red cells, leukocytes, and platelets can be studied. Rheology includes measurement of the interactions among plasma and cellular elements during flow within the microcirculation. Unfortunately, rheologic factors are generally ignored in thinking about hemodynamics. The concept of the optimal hematocrit has great clinical relevance in both acute and chronic disease. Transvascular fluid and solute exchange can be evaluated in the body as a whole and in individual organs by relatively noninvasive techniques. Such evaluations provide useful information about the natural progress and the effect of treatment of a variety of serious diseases. Microcirculation, as an entity, is a quantitative science in its own right and ought to be actively applied in clinical medicine.

It is unclear whether beta-adrenergic agonists or antagonists affect lung liquid and protein exchange by changing pulmonary hemodynamics or microvascular leakiness. In 23 unanesthetized, instrumented sheep with long-term lung lymph fistulas, we assessed the effect of the beta-agonist terbutaline or the beta-antagonists propranolol, nadolol, and atenolol, all infused intravenously, on lung lymph flow under base-line conditions and during the acute lung injury caused by 4 h of venous air embolism. Under base-line conditions, neither beta-stimulation nor blockade had any effect. During air embolism, terbutaline decreased pulmonary vascular resistance and lymph flow by 25%. Propranolol and nadolol (non-selective beta 1,beta 2-antagonists) but not atenolol (selective beta 1-antagonist) also decreased lymph flow by 22% on average. We favor the more conservative (hemodynamic) over the more liberal (altered permeability) explanation for our results. First, beta-stimulation clearly caused vasodilation, which lowered the pulmonary microvascular pressure at the site of injury. beta-blockade caused changes similar to alpha-stimulation (J. Appl. Physiol. 62: 2147-2153, 1987). We therefore interpret the beta-blockade as unmasking pulmonary arterial alpha-receptors stimulated by the air-embolism injury, thus allowing vasoconstriction upstream to the site of injury. We do not believe the explanation of the beta-agent effects requires any modulation of lung microvascular leakiness by beta-adrenergic agents.

We determined a rate of protein egress from the pleural cavity into thoracic duct lymph in seven anesthetized sheep in whom we had made 10-ml/kg hydrothoraces containing 1% plasma protein labeled with 125I-albumin. The labeled protein left the pleural space at an average rate of 0.02 ml X kg-1 X h-1. In 25 unanesthetized sheep we injected labeled protein intravenously and collected pleural fluid and plasma at intervals up to 48 h. The radioactivity in the sheeps' plasma fell twice as rapidly as the rate observed in humans. The half time for equilibration between plasma and pleural fluid protein specific activity was approximately 6 h. In seven sheep we gave a continuous infusion of tracer protein. In six of these seven sheep we collected superficial cervical lymph. The protein concentration in the lymph was three times that of pleural fluid. The half time for protein specific activity equilibration between plasma and pleural fluid was 5 h and 3-4 h between plasma and superficial cervical lymph. The rate of protein turnover in the pleural space appears to be much slower than previously reported.

To test whether platelets are necessary for the increased vascular permeability associated with microemboli, we used 16 anesthetized sheep in which we measured lung lymph flow, pulmonary arterial and left atrial pressures, thermodilution cardiac output, and lymph/plasma protein concentration. Injecting glass bead microemboli (200 micrometers diam) until pulmonary vascular resistance increased to three times base-line values caused lung lymph flow to increase at nearly constant lymph-to-plasma protein concentration ratio that is characteristic of increased microvascular permeability. Antiplatelet serum alone caused transient increases in pulmonary vascular resitance and lung lymph flow, but produced no change in steady-state lung fluid balance. After depleting platelets by greater than 97%, tripling pulmonary vascular resistance with emboli resulted in increases in lung lymph and protein flow comparable to that seen in untreated sheep. We injected twice the amount of beads in thrombocytopenic sheep compared to untreated sheep. We conclude that, although platelets do augment the pulmonary hypertension after emboli, they are not essential for the microemboli vascular injury.

Microvascular pressures in the pulmonary circulation were measured under the pleural surface of the isolated perfused dog lung by the servo-null technique. Strong glass micropipettes with short beveled tips were used, with a suction ring to stabilize the lung's surface. Of the total vascular resistance, 45 percent was in the alveolar wall capillaries themselves. Most of the remaining resistance was in the arterioles. There was negligible pressure drop in venules with diameters larger than 20 micrometers.

To determine the lung microvascular pressure profile during hypoxia, we micropunctured the subpleural microcirculation of isolated perfused cat lungs. Our procedures involved exsanguinating a cat, then cannulating its pulmonary artery, left atrium, and trachea. Using the cat's own blood, we perfused the lungs at pulmonary artery and left atrial pressures of 18 and 9 cm water, respectively, to obtain lung blood flow of 81 +/- 29 ml/(kg body weight x min), which we held constant throughout the experiment. We stabilized the lung surface with a vacuum ring and micropunctured 30- to 50-micrometers arterioles and venules to measure microvascular pressure by the servo-null method. During micropuncture, we held the lungs at constant inflation using airway pressure of 8 cm water. We varied the oxygen concentration of the inflation gas from 30% during baseline to 2% during hypoxia. We studied groups with high (more than 7.5) or normal pH. During normoxia, 27, 44, and 29% of the pressure drop occurred in the arterial, capillary and venous segments, respectively. During hypoxia, the increase in pulmonary vascular resistance, which was marked in both groups, was significantly greater in the normal pH group. All segmental pressure drops increased significantly during hypoxia. However, the predominant increase occurred in the arteries where segmental pressure drop increased by 148% and 210%, respectively, in the high and normal pH groups. We conclude that the major site of hypoxic vasoconstriction is in the pulmonary arteries.

Overperfusion (high pressure and flow through a restricted microvascular bed) has been suggested as the mechanism for both microembolic and high altitude pulmonary edema. In eighteen anesthetized, ventilated sheep, we measured pulmonary hemodynamics, lung lymph flow, and lymph:plasma protein concentration ratio. After a 2-hour stable baseline, we resected 65% of lung mass (right lung and left upper lobe) and gave whole blood transfusions to maintain cardiac output. During overperfusion of the left lower lobe, lymph flow increased moderately (5.8 +/- 2.3 to 7.7 +/- 3.8 ml/hr) and lymph:plasma protein concentration decreased (0.73 +/- 0.08 to 0.64 +/- 0.08). After a 2-hour stable period, we decreased inspired oxygen in 10 sheep (Pao2 = 40 +/- 3 mm Hg). With added alveolar hypoxia, pulmonary artery pressure increased modestly, but lymph flow and the lymph:plasma protein concentration ratio did not change. In eight sheep (four hypoxic, four normoxic), we raised left atrial pressure approximately 12 cm H2O for 2 hours. Lymph flow rose (10.8 +/- 3.8 ml/h) and lymph:plasma protein concentration decreased further (0.52 +/- 0.07). At each step, lymph:plasma protein concentration decreased, as predicted for the calculated rise in microvascular pressure. There was no evidence that overperfusion, with or without alveolar hypoxia, increased lung endothelial barrier protein permeability.

Research on lung fluid balance and pulmonary edema has increased during the last decade. New approaches have led to insights into the role of each component of the alveolar-capillary barrier. The capillary endothelium is the first line of defense against lung fluid accumulation. The interstitium may play a more important role in lung fluid balance than previously appreciated. Active and passive transport properties of alveolar epithelium may be important in the pathogenesis and resolution of alveolar edema. New methods for the determination of epithelial permeability and lung water are being evaluated. The developments reviewed here may have an impact on the institution of new diagnostic and therapeutic approaches to pulmonary edema during the next decade.

Thirty-five investigators representing the fields of anatomy, anesthesiology, biophysics, chest medicine, nuclear medicine, pathology, physiology, radiology, and surgery met to discuss the current status and future direction of the measurement of extravascular lung water. This report was prepared by the co-organizers and reviewed by the four session chairmen.

We measured the protein composition of plasma and lung fluids from nine dogs with cardiogenic edema. To produce the edema, we increased left atrial pressure an average of 36 cmH2O by inflating a balloon catheter in the left atrium, infusing norepinephrine, and infusing large volumes of saline or saline-diluted blood. Blood samples were collected every 15 min, and airway fluid was collected from five dogs that developed severe edema. Terminally, the chest was opened and the lungs were removed. Samples of alveolar fluid were taken from the excised lung by direct pleural puncture with micropipettes. The lungs were frozen in liquid nitrogen and samples of free interstitial fluid were taken from the perivascular and peribronchial cuffs. Plasma and lung fluids were analyzed for total protein by the Lowry method and for albumin-globulin fractions by cellulose acetate electrophoresis. The average total protein concentrations (g/100 ml) were--plasma, 2.65; free interstitial fluid, 1.05; alveolar fluid, 1.23; and airway fluid, 1.29. The average albumin fraction of plasma was 0.40; of alveolar fluid, 0.43; and of airway fluid, 0.43. The protein concentrations in the three lung fluids were nearly identical to each other, but were less than half that of plasma. We conclude that in high-pressure pulmonary edema with alveolar flooding, the capillary endothelium retains the ability to restrict protein relative to fluid filtration, but the alveolar epithelium becomes freely permeable to protein.

We studied anesthetized sheep to determine the relationship between increased permeability pulmonary edema and the development and mechanism of pleural effusion formation. In 12 sheep with intact, closed thoraces, we studied the time course of pleural liquid formation after 0.12 ml/kg i.v. oleic acid. After 1 h, there were no pleural effusions, even though extravascular lung water increased 50% to 6.0 +/- 0.7 g/g dry lung. By 3 h pleural effusions had formed, they reached a maximum at 5 h (48.5 +/- 16.9 ml/thorax), and at 8 h there was no additional accumulation of pleural liquid (45.5 +/- 16.9 ml). Morphologic studies by light and electron microscopy demonstrated subpleural edema but no detectable injury to the visceral pleura, suggesting that the pleural liquid originated from the lung and not the pleura. In nine sheep, we quantified the rate of formation of pleural liquid by enclosing one lung in a plastic bag. By comparing in the same sheep the volume of pleural liquid collected from the enclosed lung to the volume found in the opposite intact chest, we estimated the rate of liquid absorption from the intact chest to be 0.32 ml/(kg.h); we had previously reported a liquid absorption rate of 0.28 ml/(kg.h) in normal sheep. These studies also supported the conclusion that the majority of the pleural liquid originated from the lung because we could account for all of the pleural liquid that was formed and cleared. The volume of pleural liquid collected from the enclosed lungs was equal to 21% of the excess lung liquid that formed after oleic acid-induced lung injury. Thus, the pleural space and parietal pleural lymphatic pathways are important pathways for the clearance of pulmonary edema liquid after experimentally induced increased permeability pulmonary edema.

To determine the leakiness to protein of the barrier that separates the air space and interstitial compartment of the lung, we measured perivascular interstitial fluid cuff protein concentration and volume in 10 isolated and 9 intact closed-chest dog lung lobes, which we degassed and inflated to 25, 50, 75, or 100% of capacity with 5% bovine serum albumin labeled with Evans blue dye. After 1 h we froze the lobes in liquid N2 and made color transparencies of 20 randomly selected frozen samples of each lobe. We measured Evans blue dye-albumin concentrations from absorption by cuff images of a 50-micron-diam red (lambda = 620 nm) microspot. We measured absolute cuff volume (ml/g dry lung) by point counting on the transparencies. Using specific Evans blue-albumin fluorescence we determined that the dye was protein bound in airways and cuffs. Cuff protein concentration averaged 37% of instillate concentration and did not vary with inflation volume or between isolated and intact lobes. Cuff volume was 3.4 ml/g dry lung at total lung capacity in both isolated and intact lobes. We conclude that at some point the barrier is permeable to albumin as well as liquid at all lung volumes in dogs and that the protein sieving properties of the barrier do not change with lung expansion over the range examined. The liquid storage capacity of the cuffs can increase as much as 20-fold between low and high lung volumes.