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Obstructive sleep apnea is associated with significant cardiovascular disease, yet little is known about the effects of OSA on pulmonary microvascular perfusion. In a recent report, we showed that pulmonary microvascular perfusion was significantly mal-distributed in anesthetized, spontaneously breathing rats exposed to five episodes of obstructive apnea. We quantified microvascular perfusion by analyzing trapping patterns of 4 μm diameter fluorescent latex particles infused into the pulmonary circulation after the last episode. We could not determine if the perfusion maldistribution was due to the effects of large subatmospheric intrapleural pressures during apnea, or to precapillary OSA hypoxic vasoconstriction. To address this, we repeated these studies using isolated, buffer-perfused rat lungs (P pulm art , 10 cm H 2 O) ventilated in a chamber (-5 to -15 cm H 2 O, 25 breaths/min; P trachea  = 0). We simulated apnea by clamping the trachea and cycling the chamber pressures between -25 and -35 cm H 2 O for five breaths. After five apnea episodes, we infused 4 μm diam. fluorescent latex particles into the pulmonary artery. The number of particles recovered from the venous effluent was 74% greater in nonapneic isolated lungs compared to apneic lungs (P ≤ 0.05). Apneic lungs also had perfusion maldistributions that were 73% greater than those without apnea (P ≤ 0.05). We conclude that simulated apnea in isolated, perfused rat lungs produces significantly greater particle trapping and microvascular perfusion maldistribution than in nonapneic isolated lungs. We believe these effects are due to the large, negative intrapleural pressures produced during apnea, and are not due to hypoxia., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Pulmonary alveolar septal capillaries receive their perfusion from a web of larger surrounding acinar vessels. Using 4 μm diam. Latex particles, we showed that positive pressure ventilation narrowed the acinar vessels as evidenced by venous 4 μm particle concentrations and perfusate flows <50% of particle concentrations in negative pressure ventilated lungs. We aimed to understand the effects of positive and negative pressure ventilation on flows of larger particles through the lung. Isolated, ventilated rat lungs (air, transpulmonary pressures of 15/5 cm H2O, 25 breaths/min) were perfused with a hetastarch solution at Ppulm art/PLA pressures of 10/0 cm H2O. Red latex 7 μm (2.5 mg, 4.8 × 10 6 ) or 15 μm (2.5 mg, 5.5 × 10 5 ) particles were infused into each lung during positive or negative pressure ventilation. An equal number of green particles was infused 20 min later. Flows through lungs infused with 7 μm and 15 μm particles were not different from flows through lungs infused with 4 μm particles (p = 0.811). Venous particle concentrations of 7 μm particles relative to infused particles were lower in positive pressure lungs (0.03 ± 0.03%) compared to negative pressure lungs (0.17 ± 0.12%) (p = 0.041). Venous particle concentrations of 15 μm particles were not different between positive (2.3 ± 1.9%) and negative (3.3 ± 1.8%) pressure ventilation (p = 0.406). Together with our previous study, we conclude that 4 μm and 7 μm particles both enter acinar vessels but that the 7 μm particles are too large to flow through those vessels. In contrast, we conclude that 15 μm particles bypass the acinar vessels, flowing instead through larger intrapulmonary vessels to enter the venous outflow. These findings suggest that intrapulmonary vessels are organized as a web that allows bypass of the acinar vessels by large particles and, that these bypass vessels are not compressed by positive pressure ventilation., (Copyright © 2018. Published by Elsevier Inc.)

Obstructive sleep apnea (OSA) is associated with significant cardiovascular consequences, including pulmonary hypertension, yet little is known about its effects on pulmonary microvascular perfusion. To investigate effects of OSA on pulmonary microvascular perfusion, we clamped the tracheal cannulas of anesthetized, spontaneously breathing rats to simulate obstructive apnea. The clamp remained in place for 10 breaths before it was released to allow the animals to again breathe spontaneously. We repeated this protocol every 20 s until the rat experienced a total of five apneic episodes of 10 breaths each. We then infused into a femoral vein 10 8 fluorescent latex particles (4 µm diameter), which became trapped within the pulmonary microcirculation. We removed the lungs, allowed them to air-dry, and quantified the particle distributions in sections of the lungs using dispersion index (DI) analysis, a method we developed previously. The log of the DI (logDI) is a measure of perfusion maldistribution. Greater log(DI) values correspond to greater maldistribution. Apneic lungs had average logDI values of 1.28 (SD 0.24). Rats not subjected to apnea had average logDI values of 0.85 (SD 0.08) ( P ≤ 0.05). Rats that received latex particles 10 min or 24 h after apnea had average logDI values of 0.97 (SD 0.31) and 0.84  (SD 0.38), respectively (not significant). Our results demonstrate, for the first time, that a few apneic events produced significant, but temporary, perfusion maldistribution within the pulmonary microcirculation. Repeated nightly episodes of apnea over months and years may explain why human patients with OSA suffer from significantly greater cardiovascular disease than those without OSA.

We compared acinar perfusion in isolated rat lungs ventilated using positive or negative pressures. The lungs were ventilated with air at transpulmomary pressures of 15/5 cm H 2 O, at 25 breaths/min, and perfused with a hetastarch solution at P pulm art /P LA pressures of 10/0 cm H 2 O. We evaluated overall perfusability from perfusate flows, and from the venous concentrations of 4-µm diameter fluorescent latex particles infused into the pulmonary circulation during perfusion. We measured perfusion distribution from the trapping patterns of those particles within the lung. We infused approximately 9 million red fluorescent particles into each lung, followed 20 min later by an infusion of an equal number of green particles. In positive pressure lungs, 94.7 ± 2.4% of the infused particles remained trapped within the lungs, compared to 86.8 ± 5.6% in negative pressure lungs ( P ≤ 0.05). Perfusate flows averaged 2.5 ± 0.1 mL/min in lungs ventilated with positive pressures, compared to 5.6 ± 01 mL/min in lungs ventilated with negative pressures ( P ≤ 0.05). Particle infusions had little effect on perfusate flows. In confocal images of dried sections of each lung, red and green particles were co-localized in clusters in positive pressure lungs, suggesting that acinar vessels that lacked particles were collapsed by these pressures thereby preventing perfusion through them. Particles were more broadly and uniformly distributed in negative pressure lungs, suggesting that perfusion in these lungs was also more uniformly distributed. Our results suggest that the acinar circulation is organized as a web, and further suggest that portions of this web are collapsed by positive pressure ventilation.

Several studies have suggested that large-diameter (>25 μm) arterio-venous shunt pathways exist in the lungs of rats, dogs, and humans. We investigated the nature of these pathways by infusing specific-diameter fluorescent latex particles (4, 7, 15, 30, or 50 μm) into isolated, ventilated rat lungs perfused at constant pressure. All lungs received the same mass of latex (5 mg), which resulted in infused particle numbers that ranged from 1.7 × 10 7 4 μm particles to 7.5 × 10 4 50 μm particles. Particles were infused over 2 min. We used a flow cytometer to count particle appearances in venous effluent samples collected every 0.5 min for 12 min from the start of particle infusion. Cumulative percentages of infused particles that appeared in the samples averaged 3.17 ± 2.46% for 4 μm diameter particles, but ranged from 0.01% to 0.17% for larger particles. Appearances of 4 μm particles followed a rapid upslope beginning at 30 sec followed by a more gradual downslope that lasted for up to 12 min. All other particle diameters also began to appear at 30 sec, but followed highly irregular time courses. Infusion of 7 and 15 μm particles caused transient but significant perfusate flow reductions, while infusion of all other diameters caused insignificant reductions in flow. We conclude that small numbers of bypass vessels exist that can accommodate particle diameters of 7-to-50 μm. We further conclude that our 4 μm particle data are consistent with a well-developed network of serial and parallel perfusion pathways at the acinar level., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

We showed previously that a 30% blood loss in rats, without resuscitation, caused significant accumulation of microthrombi and leukocytes within the pulmonary circulation by 24 h. We hypothesized that the microthrombi formed spontaneously as a consequence of hemorrhage-induced stasis within the low-pressure pulmonary circuit and that the leukocytes were attracted to them. This suggested that elimination of the microthrombi, using an inhaled thrombolytic agent, could prevent the neutrophil sequestration after blood loss. To test this hypothesis, we removed 30% of the calculated blood volume from isoflurane-anesthetized, male Sprague-Dawley rats (350-500 g) over 5 min and allowed them to recover. Six hours later, we re-anesthetized the rats and nebulized tissue plasminogen activator (80 or 320 µg/kg), lactated Ringer's solution (LRS), or ipratropium bromide (i-bromide) into their lungs. We used i-bromide as a control after we discovered that nebulized LRS had thrombolytic properties. At 24 h, we removed and fixed the lungs and prepared sections for immunohistochemistry using antibodies against fibrinogen (microthrombi) and CD16 (leukocytes). Digital images of each section were obtained using a confocal microscope. Pixel counts of the images showed significantly less accumulation of microthrombi and leukocytes in lungs nebulized with tissue plasminogen activator or LRS than in non-nebulized lungs or in lungs nebulized with i-bromide (P ≤ 0.05). Lactated Ringer's solution becomes positively charged when nebulized (unlike i-bromide), suggesting that it eliminated microthrombi by fibrin depolymerization. We confirmed this using an in vitro assay. Our results demonstrate that lyses of microthrombi that accumulate in the lung after acute blood loss prevent subsequent leukocyte sequestration.

This article describes a method for quantifying blood flow distribution among lung alveoli. Our method is based on analysis of trapping patterns of small diameter (4 μm) fluorescent latex particles infused into lung capillaries. Trapping patterns are imaged using confocal microscopy, and the images are analyzed statistically using SAS subroutines. The resulting plots provide a quantifiable way of assessing interalveolar perfusion distribution in a way that has not previously been possible. Methods for using this technique are described, and the SAS routines are included. This technique can be an important tool for learning how this critical vascular bed performs in health and disease.

Intrapulmonary arteriovenous anastomoses (IPAVS) directly connect the arterial and venous circulations in the lung, bypassing the capillary network. Here, we used solid, latex microspheres and isolated rat lung and intact, spontaneously breathing rat models to test the hypothesis that IPAVS are recruited by alveolar hypoxia. We found that hypoxia recruits IPAVS in the intact rat, but not the isolated lung. IPAVS are at least 70 μm in the rat and, interestingly, appear to be recruited when the mixed venous Po(2) falls below 22 mmHg. These data provide evidence that large-diameter, direct arteriovenous connections exist in the lung and are recruitable by hypoxia in the intact animal.

Vasoconstrictors cause contraction of pulmonary microvascular endothelial cells in culture. We wondered if this meant that contraction of these cells in situ caused active control of microvascular perfusion. If true, it would mean that pulmonary microvessels were not simply passive tubes and that control of pulmonary microvascular perfusion was not mainly due to the contraction and dilation of arterioles. To test this idea, we vasoconstricted isolated perfused rat lungs with angiotensin II, bradykinin, serotonin, or U46619 (a thromboxane analog) at concentrations that produced equal flows. We also perfused matched-flow controls. We then infused a bolus of 3 μm diameter particles into each lung and measured the rate of appearance of the particles in the venous effluent. We also measured microscopic trapping patterns of particles retained within each lung. Thirty seconds after particle infusion, venous particle concentrations were significantly lower (P ≤ 0.05) for lungs perfused with angiotensin II or bradykinin than for those perfused with U46619, but not significantly different from serotonin perfused lungs or matched flow controls. Microscopic clustering of particles retained within the lungs was significantly greater (P ≤ 0.05) for lungs perfused with angiotensin II, bradykinin, or serotonin, than for lungs perfused with U46619 or for matched flow controls. Our results suggest that these agents did not produce vasoconstriction by a common mechanism and support the idea that pulmonary microvessels possess a level of active control and are not simply passive exchange vessels.

To assess the effects of intra-abdominal bacteremia on lung cellular function in vivo, we used electron microscopy to quantify the uptake of 6 nm diameter, albumin-coated colloidal gold particles (overall diam. 20.8 nm) by cells in the lungs of rats made septic by the introduction of live bacteria (E.coli and B. fragilis) into their abdomens. Gold particles were instilled into the trachea 24 hr after bacteremia induction, and lungs were harvested and prepared for electron microscopy 24 hr later. Because bacteremia produces an increase in metabolism, we hypothesized that this might be associated with increased cellular uptake of these particles and also with increased permeability of the alveolar epithelial barrier to them, as bacteremia is also associated with lung injury. We quantified particle uptake by counting particle densities (particles/μm²) within type I and type II epithelial cells, capillary endothelial cells, erythrocytes and neutrophils in the lungs of five septic rats and five sham-sepsis controls. We also counted particle densities within organelles of these cells (nuclei, mitochondria, type II cell lamellar bodies) and within the alveolar interstitium. We found particles to be present within all of these compartments, although we found no differences in particle densities between bacteremic rats and sham-sepsis controls. Our results suggest that these 6 nm particles were able to freely cross cell and organelle membranes, and further suggest that this ability was not altered by bacteremia., (Copyright © 2010 Wiley-Liss, Inc.)

We showed previously that acute blood loss, without resuscitation, caused marked maldistribution of interalveolar perfusion. Because hemorrhage is a known risk factor for the development of lung injury, the goal of our present studies was to determine if there was a correlation between perfusion maldistribution and the subsequent development of lung injury after blood loss. Specifically, we wanted to know if the perfusion maldistribution might be due to microthrombus formation and/or leukocyte sequestration within the pulmonary microcirculation. We bled rats (30% blood loss) and harvested their lungs 45 min or 24 h later. Lungs were prepared for perfusion distribution analysis, Western blot analysis to measure whole-lung fibrinogen concentrations, and for immunohistochemistry to measure fibrin deposition and leukocyte deposition (CD16 fluorescence). Perfusion was significantly maldistributed at 45 min and 24 h (P < 0.05). At 45 min, whole-lung fibrinogen concentrations were less than half that in controls (P < 0.05), whereas numbers of fibrin microthrombi were 2.5-fold greater than control by 45 min (not statistically significant) and were 4.5-fold greater by 24 h (P = 0.01). Leukocyte deposition was two-fold greater than control by 45 min (not statistically significant) and was 4-fold greater by 24 h (P = 0.02). Fibrin-to-leukocyte nearest-neighbor distances remained unchanged (18.1 [SD, 1.1] μm) even as the numbers of both increased with time after blood loss. Our results suggest that soluble fibrinogen polymerized to insoluble fibrin within minutes after acute blood loss, which caused perfusion maldistribution and attracted leukocytes. The development of lung injury after blood loss may be a consequence of leukocyte chemoattraction to fibrin microthrombi that seem to form within minutes after blood loss.

Acute hemorrhage is often followed by devastating lung injury. However, why blood loss should lead to lung injury is not known. One possibility is that hemorrhage rapidly disturbs the distribution of microvascular perfusion at the alveolar level, which may be a triggering event for subsequent injury. We showed previously that a 30% blood loss in rats caused significant maldistribution of interalveolar perfusion within 45 min (J Trauma 60:158, 2006). In this report, we describe results of further exploration of this phenomenon. We wanted to know if perfusion distribution was disturbed at 15 min, when vascular pressures were significantly reduced by the blood loss, compared with those at 45 min, when the pressures had returned substantially toward normal. We hemorrhaged rats by removing 30% of their blood volume. We quantified interalveolar perfusion distribution by statistically analyzing the trapping patterns of 4-microm-diameter fluorescent latex particles infused into the pulmonary circulation 15 (red particles) and 45 min (green particles) after blood removal. We used confocal fluorescence microscopy to digitally image the trapping patterns in sections of the air-dried lungs and used pattern analysis to quantify the patterns in tissue image volumes that ranged from 1,300 alveoli to less than 1 alveolus. LogDI, a measure of perfusion maldistribution, increased from 1.00 +/- 0.15 at 15 min after blood loss to 1.62 +/- 0.24 at 45 min (P < 0.001). These values were 0.86 +/- 0.22 (15 min) and 1.12 +/- 0.24 (45 min) in control rats (P = 0.03). Hemorrhage caused the green (45 min)-to-red (15 min) particle distance to decrease from 35.9 +/- 6.5 to 28.0 +/- 5.1 microm (P = 0.024) and the red-to-green particle distance to remain unchanged (30.2 +/- 5.7 microm [red]; 31.5 +/- 10.0 microm [green] [n.s.]). We conclude that hemorrhage caused a progressive increase in interalveolar perfusion maldistribution over 45 min that did not correspond to reduced arterial pressures or altered blood gases. Our particle distance measurements led us to further conclude that this maldistribution occurred in areas that were perfused at 15 min rather than in previously unperfused areas .

Effects of hypoxic vasoconstriction on inter-alveolar perfusion distribution (< or =1000 alveoli) have not been studied. To address this, we measured inter-alveolar perfusion distribution in the lungs of unanesthetized rats breathing 10% O(2). Perfusion distributions were measured by analyzing the trapping patterns of 4 microm diameter fluorescent latex particles infused into the pulmonary circulation. The trapping patterns were statistically quantified in confocal images of the dried lungs. Trapping patterns were measured in lung volumes that ranged between less than 1 and 1300 alveoli, and were expressed as the log of the dispersion index (logDI). A uniform (statistically random) perfusion distribution corresponds to a logDI value of zero. The more this value exceeds zero, the more the distribution is clustered (non-random). At the largest tissue volume (1300 alveoli) logDI reached a maximum value of 0.68+/-0.42 (mean+/-s.d.) in hypoxic rats (n = 6), 0.50+/-0.38 in hypercapnic rats (n.s.) and 0.48+/-0.25 in air-breathing controls (n.s.). Our results suggest that acute hypoxia did not cause significant changes in inter-alveolar perfusion distribution in unanesthetized, spontaneously breathing rats.

Objective: Sepsis often leads to lung injury, although the mechanisms that initiate this are unclear. One preinjury phenomenon that has not been explored previously is the effect of bacterial (nonlipopolysaccharide) sepsis on the distribution of alveolar perfusion. The goals of our studies were to measure this., Design: Randomized, controlled, prospective animal study., Setting: University animal laboratory., Subjects: Male Sprague-Dawley rats (450-550 g)., Interventions: We induced sepsis by placing gelatin capsules containing Escherichia coli and Bacteroides fragilis into the abdomens of rats (n = 9). Empty capsules (n = 6) were placed into the abdomens of controls. After 24 hrs, 4-microm-diameter fluorescent latex particles (2 x 10(8)) were infused into the pulmonary circulation. Sepsis was induced in additional rats and controls to assess lung injury, as follows: Lung histology was performed on eight septic rats and on seven controls; lung lavage was performed on three septic rats and three controls after their plasma albumin had been labeled with Evans blue dye., Measurements and Main Results: Confocal microscopy was used to prepare digital maps of latex particle trapping patterns (eight per lung). Analysis of these patterns revealed statistically more clustering (perfusion inhomogeneity) down to tissue volumes less than that of ten alveoli in septic lungs compared with controls (p < or = .05). Bacterial counts and neutrophil counts were significantly higher in the circulation of septic rats (p < or = .05). Blood pressures and arterial PO2s were unchanged. Cell counts in histological images were three-fold higher in septic lungs than in controls (p < or = .05). Lung lavage revealed 0.41 +/- 0.03 mL of plasma in the lungs of septic rats, and 0.06 +/- 0.05 mL in the lungs of controls (p < or = .05)., Conclusions: Bacterial sepsis caused significant maldistribution of interalveolar perfusion in the lungs of rats in the absence of significant lung injury.

Background: Lung injury often occurs following hemorrhage and we hypothesized that this might be due to the effects of hemorrhage on perfusion distribution among alveoli. To test this, we measured interalveolar perfusion distribution in anesthetized, spontaneously breathing rats subjected to blood losses of 0%, 10%, 20%, or 30% of calculated blood volume., Methods: We measured interalveolar perfusion distribution by analyzing trapping patterns of 4-mum diameter fluorescent latex particles infused into the pulmonary circulation. The particles (2 x 10) were infused 1 hour after each animal had been bled, and the lungs were then removed and air-dried. Using a confocal fluorescence microscope, we collected images of the particles in eight sections of each lung. Each image encompassed 3,360 x 3,360 x 100 microm (approximately 5,000 alveoli), and included 3-4,000 particles. Particle distributions in the images were measured using the method of dispersion index (DI) analysis. A DI value of zero corresponds to a statistically random distribution; the more DI exceeds zero, the more the distribution is clustered or inhomogenous., Results: The largest DI values for the four groups were: 0%, 0.69 +/- 0.41; 10%, 0.57 +/- 0.58; 20%, 0.72 +/- 0.34; 30%, 1.38 +/- 0.41. The 30% blood loss group had a max DI value approximately twofold greater than those of the other three (p < 0.0001)., Conclusions: Our results suggest that interalveolar perfusion distribution becomes markedly maldistributed at blood losses of 30%. This contributes to ventilation-perfusion mismatching, and may be a precipitating event for lung injury following hemorrhage.

Purpose: We present preclinical data showing the in vitro intranuclear uptake of motexafin gadolinium by glioblastoma multiforme cells, which could serve as a prelude to the future development of radiosensitizing techniques, such as gadolinium synchrotron stereotactic radiotherapy (GdSSR), a new putative treatment for glioblastoma multiforme., Experimental Design: In this approach, administration of a tumor-seeking Gd-containing compound would be followed by stereotactic external beam radiotherapy with 51-keV photons from a synchrotron source. At least two criteria must be satisfied before this therapy can be established: Gd must accumulate in cancer cells and spare the normal tissue; Gd must be present in almost all the cancer cell nuclei. We address the in vitro intranuclear uptake of motexafin gadolinium in this article. We analyzed the Gd distribution with subcellular resolution in four human glioblastoma cell lines, using three independent methods: two novel synchrotron spectromicroscopic techniques and one confocal microscopy. We present in vitro evidence that the majority of the cell nuclei take up motexafin gadolinium, a drug that is known to selectively reach glioblastoma multiforme., Results: With all three methods, we found Gd in at least 90% of the cell nuclei. The results are highly reproducible across different cell lines. The present data provide evidence for further studies, with the goal of developing GdSSR, a process that will require further in vivo animal and future clinical studies.

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.

The growth rate and albumin concentration of interstitial fluid cuffs were measured in isolated rabbit lungs inflated with albumin solution (3 g/dl) to constant airway (Paw) and vascular pressures for up to 10 h. Cuff size was measured from images of frozen lung sections, and cuff albumin concentration (Cc) was measured from the fluorescence of Evans blue labeled albumin that entered the cuffs from the alveolar space. At 5-cmH2O Paw, cuff size peaked at 1 h and then decreased by 75% in 2 h. The decreased cuff size was consistent with an osmotic absorption into the albumin solution that filled the vascular and alveolar spaces. At 15-cmH2O Paw, cuff size peaked at 0.25 h and then remained constant. Cc rose continuously at both pressures, but was greater at the higher pressure. The increasing Cc with a constant cuff size was modeled as diffusion through epithelial pores. Initial Cc-to-airway albumin concentration ratio was 0.1 at 5-cmH2O Paw and increased to 0.3 at 15 cmH2O, a behavior that indicated an increased permeability with lung inflation. Estimated epithelial reflection coefficient was 0.9 and 0.7, and equivalent epithelial pore radii were 4.5 and 6.1 nm at 5- and 15-cmH2O Paw, respectively. The initial cuff growth occurred against an albumin colloid osmotic pressure gradient because a high interstitial resistance reduced the overall epithelial-interstitial reflection coefficient to the low value of the interstitium.

The transport properties of lung interstitium were studied by measuring the flow of hetastarch solution (2 and 6%) through 1-cm perivascular interstitial segments of rabbit lungs. Hetastarch (10(4)-10(7) Da) solution has a colloid osmotic pressure similar to that of albumin solution. Driving pressure was 5 cm H(2)O and mean interstitial pressure was 0 cm H(2)O. The flows of 2 and 6% hetastarch solutions were measured before (Q(1)) and after (Q(2)) the addition of 0.02% hyaluronidase. Hetastarch molecular distributions in effluent samples were measured by high-performance size-exclusion chromatography (HPSEC) to determine sieving ratio (C(out)/C(in), downstream-to-upstream concentration ratio). Hyaluronidase significantly (P < 0.0004) increased flow sixfold, but the increase in flow (Q(2)/Q(1)) was reduced through the interstitium around smaller vessels. A similar behavior was observed with the flow of albumin solution without and with hyaluronidase. C(out)/C(in) decreased monotonically with molecular weight, was greater with 6% than with 2% (low colloid osmotic pressure) hetastarch, and increased with hyaluronidase. Modeling the transport through uniform pores, equivalent pore radius was 10 and 15 nm with 2 and 6% hetastarch, respectively, and doubled with hyaluronidase. In conclusion, interstitial pores expand in response to an increase in colloid osmotic pressure both before and after tissue degradation by hyaluronidase.

Pulmonary vascular perfusion has been shown to follow a fractal distribution down to a resolution of 0.5 cm(3) (5E11 microm(3)). We wanted to know whether this distribution continued down to tissue volumes equivalent to that of an alveolus (2E5 microm(3)). To investigate this, we used confocal microscopy to analyze the spatial distribution of 4-microm-diameter fluorescent latex particles trapped within rat lung microvessels. Particle distributions were analyzed in tissue volumes that ranged from 1.7E2 to 2.8E8 microm(3). The analysis resulted in fractal plots that consisted of two slopes. The left slope, encompassing tissue volumes less than 7E5 microm(3), had a fractal dimension of 1.50 +/- 0.03 (random distribution). The right slope, encompassing tissue volumes greater than 7E5 microm(3), had a fractal dimension of 1.29 +/- 0.04 (nonrandom distribution). The break point at 7E5 microm(3) corresponds closely to a tissue volume equivalent to that of one alveolus. We conclude that perfusion distribution is random at tissue volumes less than that of an alveolus and nonrandom at tissue volumes greater than that of an alveolus.

To evaluate the transport properties of the alveolar epithelium, we instilled hetastarch (Het; 6%, 10 ml, 1 - 1 x 10(4) kDa) into the trachea of isolated rat lungs and then measured the molecular distribution of Het that entered the lung perfusate from the air space over 6 h. Het transport was driven by either diffusion or an oncotic gradient. Perfusate Het had a unique, bimodal molecular weight distribution, consisting of a narrow low-molecular-weight peak at 10-15 kDa (range, 5-46 kDa) and a broad high-molecular-weight band (range 46-2,000 kDa; highest at 288 kDa). We modeled the low-molecular-weight transport as (passive) restricted diffusion or osmotic flow through a small-pore system and the high-molecular-weight transport as passive transport through a large-pore system. The equivalent small-pore radius was 5.0 nm, with a distribution of 150 pores per alveolus. The equivalent large-pore radius was 17.0 nm, with a distribution of one pore per seven alveoli. The small-pore fluid conductivity (2 x 10(-5) ml. h(-1). cm(-2). mmHg(-1)) was 10-fold larger than that of the large-pore conductivity.

High lung inflation pressures compress alveolar septal capillaries, impede red cell transit, and interfere with oxygenation. However, recently introduced acellular hemoglobin solutions may enter compressed lung capillaries more easily than red blood cells. To test this hypothesis, we perfused isolated rat lungs with fluorescently labeled diaspirin cross-linked hemoglobin (DCLHb; 10%) and/ or autologous red cells (hematocrit, 20). Septal capillaries were compressed by setting lung inflation pressure above vascular pressures (zone 1). Examination by confocal microscopy showed that DCLHb was distributed throughout alveolar septa. Furthermore, this distribution was not affected by adding red blood cells to the perfusate. We estimated the maximum acellular hemoglobin mass within septa to be equivalent to that of 15 red blood cells. By comparison, we found an average of 2.7 +/- 4.6 red cells per septum in zone 1. These values increased to 30.4 +/- 25.8 and 50.4 +/- 22.1 cells per septum in zones 2 and 3, respectively. We conclude that perfusion in zone 1 with a 10% acellular hemoglobin solution may increase the hemoglobin concentration per septum up to fivefold compared with red cell perfusion.

Background: Gram-negative lipopolysaccharide (LPS) has been demonstrated to increase pulmonary capillary permeability as judged by the increased flow of protein-rich lymph from the lungs of sheep infused with LPS. This finding suggests that LPS-injured pulmonary capillaries might be less restrictive than uninjured capillaries to the filtration of large hetastarch molecules. Hetastarch has a broad molecular mass spectrum (35-1,500 kilodaltons (kDa)), and one way to test the restrictiveness of pulmonary capillaries is to measure the size of the largest hetastarch molecules that cross the microvascular barrier and enter the lymph. To evaluate the effects of LPS, we compared hetastarch molecular distributions in the lung lymph of normal and LPS-injured sheep., Methods: Adult sheep (38.2 +/- 0.8 kg) were surgically prepared for the collection of lung lymph, with study initiation after a 5- to 7-day recovery period. Hetastarch (6%) was infused (10 mL/kg) 24 hours before study to allow for stabilization of the hetastarch molecular distribution. On the day of study, LPS (Escherichia coli lipopolysaccharide, 2 microg/kg; n = 6) was infused, and plasma and lymph samples were collected for 12 hours. An additional group of animals not infused with LPS (n = 6) served as controls. Hetastarch molecular distributions in plasma and lymph were measured by using high performance size exclusion chromatography., Results: In control sheep, the largest hetastarch molecules in lymph averaged 861 +/- 18 kDa (mean +/- SEM) (plasma, 1,065 +/- 18 kDa). In LPS-treated sheep, the largest hetastarch molecules in lymph averaged 845 +/- 19 kDa (not significant vs. normal) (plasma, 1,025 +/- 14 kDa). Hetastarch concentrations in plasma and lung lymph of normal sheep, respectively, were 0.61 +/- 0.05% and 0.34 +/- 0.07%. In LPS-treated sheep, hetastarch concentrations in plasma and lymph were 0.56 +/- 0.08 (not significant vs. normal) and 0.29 +/- 0.07, respectively (p < or = 0.05). Lymph concentrations were lower after LPS because of increased lymph flows (19.9 +/- 5.4 mL/30 min, compared with 3.6 +/- 0.8 mL/30 min in normal sheep)., Conclusion: Our results suggest that LPS does not alter the diameter of the largest pores perforating the walls of pulmonary capillaries. Rather, the number of these pores in the capillary wall appears to be increased. This increase would explain why lymph flows rise after LPS with little change in the lymph protein concentration. Our results are also consistent with a filtration model in which capillaries are assumed to be perforated by small pores (protein reflection coefficient = 1) as well as large pores (protein reflection coefficient = 0).

Background: A limitation to fully using lung transplantation for patients with end-stage lung diseases is short, safe preservation time (4 to 6 hours). Our goal is to extend this to 24 hours or more, which would greatly improve clinical lung transplantation., Methods: We used the isolated perfused rat lung to test how two preservation solutions (low potassium dextran and University of Wisconsin solution) affected quality of lungs after 6, 12, and 24 hours of preservation. Also, we tested modifications of the University of Wisconsin solution, including reversing the ratio of Na/K, the addition of 1.5 mmol/L calcium, and the combination of calcium and butanedione monoxime, agents that improve cardiac preservation. After preservation at 4 degrees C, lungs were reperfused at 37 degrees C with a physiologically balanced solution. Pulmonary artery flow rate, airway peak inspiratory pressure, and tissue edema were used to assess degree of preservation and reperfusion injury., Results: Low potassium dextran solution gave poor preservation (decreased pulmonary artery flow, tissue edema) after 12 hours of cold storage. There were no differences between regular and reversed Na/K ratio University of Wisconsin solutions at 12 or 24 hours of preservation. Addition of calcium had no beneficial effect on lung preservation. However, University of Wisconsin solution with calcium and butanedione monoxime gave excellent 24-hour cold storage, with pulmonary artery flow rate, tissue edema, and airway peak inspiratory pressure equal to control (0 hours of preservation) lungs., Conclusions: The University of Wisconsin solution appears capable of lung preservation for up to 24 hours if modified to contain calcium and butanedione monoxime. The mechanism of action of butanedione monoxime may be related to the suppression of smooth muscle contraction resulting in vasodilation of the cold-stored lung on reperfusion.

Diaspirin crosslinked hemoglobin (DCHb) is a new blood substitute manufactured from human blood. To evaluate its microvascular filtration properties, we infused DCLHb into unanesthetized sheep (10%, 20 ml/kg) and measured the flow and composition of lung and soft tissue lymph. For comparison, we also infused human serum albumin (HSA; 10%, 20 ml/kg). DCLHb raised systemic and pulmonary arterial pressures from baseline values of 83 +/- 7 and 13 +/- 2 mm Hg, respectively, to peak values of 113 +/- 9 and 26 +/- 3 mm Hg (p < 0.05 versus baseline). These increases were significantly greater than those associated with HSA, which raised systemic and pulmonary arterial pressures from baseline values of 86 +/- 4 and 13 +/- 2 mm Hg, respectively, to peak values of 97 +/- 3 and 21 +/- 7 mm Hg (p <= 0.05 versus baseline and versus DCLHb). These differences reflect the known pressor properties of DCLHb. Accordingly, DCLHb raised lung and soft tissue lymph flows to peak values of 12.2 +/- 3.8 and 1.6 +/- 0.7 ml/30 min, respectively, while HSA raised lung and soft tissue lymph flows to peak values of 7.5 +/- 4.8 and 4.6 +/- 1.9 ml/30 min, respectively (p <= 0.05 versus DCLHb). The half-times of DCLHb equilibration from plasma into lung and soft tissue lymph of 1. 0 +/- 0.3 and 2.1 +/- 1.1 h, respectively, were significantly faster than HSA equilibration half-times of 3.1 +/- 0.2 and 3.8 +/- 0.9 h. Filtration differences between DCLHb and HSA appear to be due to the pressor properties DCLHb.

To estimate the functional diameter of alveolar microvessels, we perfused isolated rat lungs with fluorescent latex particles (1 diameter/lung) at inflation, pulmonary arterial, and left atrial pressures of 25, 30, and 0 cmH2O, respectively. We used confocal microscopy to count latex particles within septal microvessels and flow cytometry to count particle concentrations in venous outflow. We found 1-, 2-, and 4-micron-diameter particles within septal vessels of 45 +/- 12, 31 +/- 12, and 25 +/- 9%, respectively, of examined alveoli. Particles of 5-micron diameter were absent from septal vessels but were present within a small percentage of corner vessels. Particle concentrations in the venous outflow for 1-, 2-, 4-, and 5-micron-diameter particles were 54 +/- 28, 67 +/- 32, 2.2 +/- 0.3, and 0.4 +/- 0.3%, respectively, of the arterial inflow. Particles with diameters of 6 or 10 micron were absent from venous outflow. Our results suggest that, under these conditions, the functional diameter of the septal microvessels is approximately 4 micron and that the diameter of the adjacent corner vessels is slightly larger but <6 micron.

We used high performance size exclusion chromatography (HPSEC) to measure concentrations and molecular masses of hetastarch (Het) in plasma and lung lymph of unanesthetized sheep. Our goal was to assess the osmotic effectiveness of Het in the pulmonary circulation as judged by its exclusion from lung lymph. Sheep (n = 5) received 35 ml/kg of Het (6%) over 90 min. At the end of the infusion, Het concentrations in plasma reached a peak value of 2.9 +/- 0.1% (mean +/- SD). Lymph concentrations reached a peak value of 1.3 +/- 0.3% at 4.5 h. Het molecular masses in plasma averaged 650 +/- 36 kD at 90 min, but ranged from 31 to 2,942 +/- 187 kD. Masses in lung lymph averaged 373 +/- 71 kD, and ranged from 19 +/- 2 to 1,693 +/- 514 kD (p < or = 0.05 vs. plasma). Het contributed 6.7 +/- 1.5 mm Hg to the plasma macromolecular osmotic pressure, and 3.7 +/- 1.8 mm Hg to the lymph osmotic pressure. Despite the fact that Het has the largest molecular mass of any of the current macromolecular plasma volume expanders, we found that it filtered readily into lymph, raising the lymph osmotic pressure. These findings suggest that the rationale for the osmotic performance of such solutions may need to be reconsidered.

Objective: To measure the functional diameter of alveolar septal microvessels under conditions in which the pulmonary arterial pressure and the lung inflation pressure are equal, at 25 cm H2O (zone I-II border), and to compare these results with those obtained when inflation pressure exceeded arterial pressure by 5 or 10 cm H2O (zone l)., Methods: We perfused isolated rat lungs (PA 25, PPA 25, PLA 0 cm H2O) with fluorescent latex particles of specific diameters (0.49, 1.05, 2.0, 4.0, or 10 microns) and then prepared samples for histology. Using a confocal, laser-scanning fluorescence microscope, we measured latex particle densities within the septal plane of individual alveoli. We compared these particle densities with those in arterioles supplying the septa and calculated the density ratio. We fit curves produced by the Verniory equation to these ratios to estimate the septal microvessel functional diameter., Results: Particle densities in septa ranged from 0.06 +/- 0.02 particles per microns2 for 0.49-micron-diameter particles to 0.007 +/- 0.004 particles per microns2 for 4.0-microns-diameter particles. The 10-microns particles did not enter septa. Calculations based on these data suggest a septal microvessel functional diameter of 6-8 microns., Conclusions: In a previous study, conducted at the same value of Pinflat, but with PPA set at 15 or 20 (5 or 10 cm H2O into zone I). We estimated the capillary diameter to be 1.7 microns. Thus, the septal capillary diameter seems to increase by three- to fourfold as PPA is raised to equal Pinflat.

We tested the hypothesis that plasma oncotic pressure alone, not the plasma-to-lymph oncotic pressure difference, modulates pulmonary transvascular fluid filtration. To do this we measured lung lymph flow after raising left atrial pressure (by inflating a balloon) in sheep that were receiving a continuous (32 h) infusion of dextran 40. For comparison, we also raised left atrial pressure elevation, plasma oncotic pressures in dextran and control sheep, respectively, were 39.5 +/- 4.5 and 17.7 +/- 2.2 mm Hg; plasma-to-lymph oncotic pressure gradients, respectively, were 4.4 +/- 0.6 and 4.4 +/- 0.6 mm Hg. Left atrial pressure elevation during dextran infusion increased lung lymph flow by a factor of 2.4 +/- 0.4, compared with a factor of 4.2 +/- 2.3 in control sheep. Thus, left atrial pressure elevation increased lymph flow less in dextran-treated animals than in control animals, even though the plasma-to-lymph oncotic pressure gradients were equal. This suggests that plasma oncotic pressure alone may be a more important determinant of pulmonary transvascular fluid filtration than the plasma-to-lymph oncotic pressure difference.

Background: Pulmonary edema is a complication of critical care fluid management that may be restricted by the use of oncotically effective resuscitation fluids. Potentially beneficial oncotic properties of starch-based plasma volume expanders such as hetastarch (Het), pentafraction (Pen), and Dextran-70 (Dex) may be compromised by their broad range of molecular masses, some of which are small enough to filter from the circulation. Leakage of these molecules into the pulmonary interstitium may limit their oncotic effectiveness and enhance fluid filtration. We measured the filtration of these three resuscitation solutions into lung lymph to evaluate their oncotic contribution to pulmonary edema formation., Materials and Methods: Unanesthetized euvolemic adult sheep, prepared with chronic lung lymph fistulae, underwent plasma volume expansion with Het (n = 6), Pen (n = 6), or Dex (n = 6 ) (6%, 35 ml/kg/90 min). Oncotic effectiveness was determined by measuring plasma and lymph oncotic pressures and the oncotic pressures contributed by each starch. Pulmonary hydrostatic pressures and lung lymph flows (Q(L)) were also measured. Results are expressed as means +/- SEM. Comparisons were made by two-factor analysis of variance., Results: Dex contributed 9.0 +/- 0.9 mmHg to the plasma oncotic pressure, significantly more than Het and Pen (5.3 +/- 0.6, 6.5 +/- 0.6 mmHg, respectively). However, Dex filtration also contributed 6.1 +/- 0.5 mmHg to the lymph oncotic pressure, compared to 3.1 +/- 0.3 and 4.7 +/- 0.5 mmHg for Het and Pen, respectively (P < or = 0.05). Dex, Het, and Pen raised Q(L) over baseline by 7.7 +/- 1.5, 4.3 +/- 1.0, and 3.2 +/- 0.7 ml/30 min, respectively (P < or = 0.05). Dex increased Q(L) significantly more than Het or Pen., Conclusions: Pen and Het demonstrated greater oncotic effectiveness because of restricted plasma-to-lymph macromolecular filtration and limited transvascular fluid flux. By comparison, Dex filtered rapidly and increased transvascular fluid filtration. Pen appears to possess filtration properties that optimize critical care fluid management compared to currently available colloid solutions such as Het and Dex.

To estimate the functional diameter of alveolar septal microvessels in zone 1, we perfused isolated rat lungs with fluorescent latex particles of specific diameters (0.24, 0.49, 1.05, or 4.0 microns) at pulmonary artery pressures (Ppulmart) that were either 5 or 10 cmH2O less than the air inflation pressure (Pinflat, 25 cmH2O). We then prepared samples for histology. Using a confocal, laser-scanning fluorescence microscope, we measured latex particle densities within the septal plane that ranged from 0.08 +/- 0.04 particles/microns2 (0.24-microns diameter particles) to 0.02 +/- 0.01 particles/microns2 (1.05-microns diameter particles). We found that 4.0-microns diameter particles were not able to enter septa at all. Latex particles were not present in all alveoli When Ppulmart was 5 cmH2O less than Pinflat, 32 +/- 6% of septa contained 0.24-microns diameter particles, but, when Ppulmart was 10 cmH2O less than Pinflat, 5 +/- 6% of septa contained these particles. Percentages were smaller for larger particles. We conclude that, when Ppulmart is both 5 and 10 cmH2O less than Pinflat, the functional diameter of accessible septal microvessels is > 1.05 but < 4.0 microns. Furthermore, the number of accessible septa decreases as the difference between Ppulmart and Pinflat widens.

Macromolecules are present in lung preservation solutions to limit liquid filtration out of the pulmonary circulation and minimize pulmonary edema. We tested the effectiveness of these molecules by measuring interstitial edema in rat lungs perfused with macromolecular solutions (University of Wisconsin [UW] solution and Euro-Collins solution supplemented with modified pentastarch [pentafraction, PEN]) or with solutions that lacked macromolecules (UW solution with PEN and Euro-Collins solution.) The lungs were inflated with air and perfused with one of the test solutions, then rapidly frozen and prepared for histological analysis. From tissue sections, we measured cross-sectional areas of pulmonary arteries and veins, and also measured cross-sectional areas of the interstitial spaces surrounding arteries and veins. We then calculated the interstitium-to-vessel cross-sectional area ratio. In lungs perfused with macromolecular solutions these ratios were 0.09+/-0.15 and 0.53+/-0.56 (mean +/- SD) for UW solution and Euro-Collins solutions solution with PEN, respectively (P

Lung liquid conductance (Kf) is calculated as the quotient of lung lymph flow divided by net filtration pressure (Pnf), where Pnf is the balance of osmotic and hydrostatic pressures in the lung microcirculation. In protein depletion, lymph flow rises with little change in Pnf, suggesting that calculated Kf also rises. However, several previous reports have concluded that protein depletion causes little change in Kf, leaving open the question of how lung lymph flow can rise in protein depletion with little change in Pnf. To address this, we measured Kf in sheep following two kinds of protein depletion: batch plasmapheresis (BP; n = 5) and thoracic duct drainage (TD; n = 5). Both methods lowered plasma protein concentrations by 30%, and raised lung lymph flows by 55%. Lung microvascular hydrostatic pressures and plasma-to-lymph osmotic pressure gradients both changed by 1 to 2 mm Hg. With BP, calculated Kf rose from 0.26 +/- 0.09 at baseline to 0.50 +/- 0.20 on Day 1, and to 0.39 +/- 0.27 ml/mm Hg/30 min on Day 2 (p < or = 0.05). With TD, calculated Kf rose from 0.28 +/- 0.13 at baseline to 0.43 +/- 0.19 on Day 1, and to 0.43 +/- 0.19 ml/mm Hg/30 min on Day 2 (p < or = 0.05). Calculated Kf rose because filtration increased even though the hydrostatic and osmotic driving forces responsible for filtration changed little. This is puzzling because it suggests that lymph flow rose with little or no change in the forces affecting filtration. Our findings contradict several previous reports that concluded that protein depletion produces little or no change in calculated Kf.

Purpose of Study: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky., Methods: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated., Results: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05)., Conclusions: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.

Background: Hetastarch and pentafraction are high molecular weight starch solutions designed to augment plasma oncotic pressure. Although clinical utilization of hetastarch has been limited by reported coagulation abnormalities, pentafraction is a newer derivative that appears to have few adverse hemostatic effects. We examined the ability of pentafraction to modulate lung and soft tissue transvascular fluid filtration under hypoproteinemic conditions compared with hetastarch and Ringer's lactate (LR)., Methods: Awake, protein-depleted sheep (n = 19) were prepared with lung and soft tissue lymph fistulas, and comparable infusions of 5% pentafraction (n = 6), 6% hetastarch (n = 6), or LR (n = 7) were administered. Plasma and lymph samples were collected during 24-hour period to determine changes in protein concentrations, plasma-to-lymph oncotic gradients, and lung (QL) and soft tissue (QS) lymph flows., Results: QL and QS rose nearly twofold after protein depletion alone. LR infusion increased QL and QS to 8.7 +/- 1.7 and 3.1 +/- 0.6 times normoproteinemic baseline, respectively (p < 0.05). In contrast, hetastarch and pentafraction infusion limited the increase in QL to 4.2 +/- 1.1 and 4.0 +/- 0.8 times normoproteinemic baseline, respectively (p < 0.05 versus LR) and did not significantly increase QS. Hetastarch and pentafraction infusions increase plasma oncotic pressure by nearly 6 mm Hg, which significantly widened the plasma-to-lymph oncotic pressure gradients above preinfusion baseline by 4.7 +/- 0.7 and 3.4 +/- 0.4 mm Hg in lung and 4.6 +/- 0.7 and 3.2 +/- 0.4 mm Hg in soft tissue, respectively (p < 0.05)., Conclusions: Both hetastarch and pentafraction limit transvascular fluid filtration under hypoproteinemic conditions by augmenting plasma oncotic pressure and the plasma-to-lymph oncotic pressure gradient. Because of fewer adverse hemostatic effects pentafraction may be an improvement over current therapies in critical care fluid management.

Effects of infusing pentafraction (Pen), a synthetic hydroxyethyl starch plasma volume expander, on lung and soft tissue lymph flux were compared in nonanesthetized sheep that were protein depleted by batch plasmapheresis. Pen (5%) was infused to raise pulmonary arterial wedge pressure by 5 mmHg for 2 h (1.8 +/- 0.3 l). Pen raised plasma osmotic pressure from plasmapheresis baseline (10.7 +/- 2.2 mmHg; preplasmapheresis baseline, 19.6 +/- 0.6 mmHg) to 16.6 +/- 2.4 mmHg. After Pen, lung lymph flows peaked at 3.9 +/- 2.0 times a preplasmapheresis baseline value of 1.0 (plasmapheresis baseline, 2.7 +/- 0.7), but soft tissue lymph flows rose insignificantly. Plasma Pen concentrations were 2.3 +/- 1.0% postinfusion and 1.6 +/- 0.3% at 12 h. Pen mean molecular masses at these times, measured by high-performance liquid chromatography, were 160 +/- 44 and 129 +/- 23 kDa, respectively. In lung lymph, Pen concentrations were 0.8 +/- 0.6% postinfusion and 0.7 +/- 0.2% at 12 h, with mean molecular masses of 125 +/- 44 and 112 +/- 18 kDa, respectively. In soft tissue lymph Pen was nearly undetectable postinfusion, but at 12 h concentrations averaged 0.3 +/- 0.2% with a mean molecular mass of 80 +/- 10 kDa. The osmotic effectiveness of Pen may be related to its molecular mass, which was large enough to restrict filtration so that the plasma-to-lung lymph osmotic pressure gradient widened. Pen remained effective in the circulation for at least 24 h.

The combination of high inflation and low vascular pressures in zone 1 lungs is assumed to collapse alveolar vessels, making them inaccessible to vascular liquid. To test this assumption, we perfused isolated rat lungs in zone 1 (n = 5) with fluorescent albumin solution (inflation pressure = 25 cmH2O, pulmonary arterial pressure = 10 cmH2O, left atrial pressure = 0 cmH2O; flow = 0.11 +/- 0.06 ml.100 g body wt-1 x min-1) and rapidly froze them. Histologically, 33 +/- 19% (SD) of alveolar septa fluoresced, demonstrating that the perfusate had not been excluded. However, we could not resolve whether the fluorescence originated in the septal microvascular lumen or in the adjacent perimicrovascular interstitial space. To address this issue, we perfused an additional lung with horseradish peroxidase (HRP) and examined it by transmission electron microscopy. HRP filled interstitial spaces around septal vessels and extraseptal alveolar corner vessels, but because the septal vascular lumina were too compressed, we were unable to determine whether they also contained HRP. Therefore we perfused two additional lungs with particles of colloidal gold (0.05 microns diam). Using transmission electron microscopy, we found gold particles in 15-25% of septal vascular lumina, demonstrating that septal vessels were at least partially accessible in zone 1. Our interpretations is that filtration in zone 1 may occur from septal vessels and extraseptal alveolar vessels. Furthermore, results of the HRP study suggest that the perimicrovascular interstitial space is less compressible than the septal vascular lumen.

We used a simplified two-pore filtration model to examine the effects of hypoproteinemia on lung and soft tissue lymph flux in awake sheep (n = 7). To induce hypoproteinemia, we subjected each animal to 3 days of batch plasmapheresis (6 units per day). Data were collected in near steady-state conditions, 15-18 hr following completion of the last plasmapheresis episode. At this time, plasma protein concentration had fallen by 34%, while lung and soft tissue lymph protein concentrations had fallen by 55 and 62%, respectively. Lung and soft tissue lymph flows increased 52 and 87%, respectively. The plasma-to-lymph osmotic pressure gradients for lung and soft tissue lymph were unchanged by protein depletion (soft tissue, 7.7 mm Hg; lung, 4.8 mm Hg). We applied these results to a heteropore model of the microvascular barrier that consisted of two types of pores: those which plasma proteins could not cross (sigma = 1) and those which proteins could cross without restriction (sigma = 0). We varied the proportion of small pores to large pores until the measured data fit a model in which the calculated microvascular hydrostatic pressures in normal and hypoproteinemic conditions were equal. This was based on the assumption that microvascular hydrostatic pressure did not change with plasma protein depletion. These conditions could be satisfied when the small pores accounted for 90% of total barrier porosity. According to the model, lymph flow increased in hypoproteinemia because of an increase in protein-free liquid flux through the large percentage of small pores; protein flux through the small percentage of large pores remained unchanged. The net result was an increase in lymph flow and a decrease in the lymph protein concentration. The model reproduced these changes even though the plasma-to-lymph osmotic pressure gradients were unchanged. We conclude that a simplified heteropore model can explain the effects of hypoproteinemia on lung and soft tissue lymph flux.

The sequential pattern of perivascular interstitial cuff growth was studied in liquid-inflated rabbit lungs. Degassed isolated lungs were immersed in a saline bath and inflated to 5 cmH2O transpulmonary pressure with a 3% albumin solution or 3% albumin solution containing hyaluronidase. After inflation times varying between 1 and 7 h, the lungs were frozen in liquid N2. From blocks cut from the frozen lungs, interstitial cuff cross-sectional area was measured as a function of vessel size. No cuffs were observed around vessels less than 0.1 mm diam. At all inflation times, only approximately 50% of vessels less than 0.5 mm diam had cuffs, whereas virtually all vessels greater than 0.5 mm diam had cuffs. Cuff-to-vessel area ratio increased with inflation time, reaching a maximum of 1.0-1.4 by 5 h. The time constant of cuff growth was 1 h for the albumin-inflated lungs and was independent of vessel size. The time constant was reduced by 60% in the hyaluronidase-inflated lungs. The time constant for the response in perivascular interstitial pressure measured by micropuncture near the lung hilum was 2.5 h for albumin-inflated lungs and 1.2 h for hyaluronidase-inflated lungs. Electrical analog models were used to fit the experimental data of cuff growth and to determine interstitial liquid resistance. Interstitial resistance for the albumin-inflated rabbit lungs was 2- and 24-fold greater than values estimated previously for sheep and dog lungs, respectively.

Interleukin-2 has been widely investigated as adjuvant therapy for advanced cancer and is administered by either bolus or continuous infusion. We compared the effects of bolus and continuous interleukin-2 infusion on pulmonary (QL) and systemic microvascular fluid filtration in 11 adult sheep prepared with chronic lung and soft-tissue lymph fistulas. Interleukin-2 was administered as a bolus infusion (100,000 units/kg) every 8 hours for 3 days or as a continuous infusion at the same dose for 3 days. No significant changes in pulmonary hydrostatic pressures or pulmonary vascular resistance were noted after either bolus or continuous interleukin-2 infusion. However, significantly decreased (p less than or equal to 0.05) systemic vascular resistances were observed in both groups. QL increased steadily throughout the infusion period in both groups, peaking at three times baseline on the third infusion day. The plasma/interstitial protein clearance (QL X lymph/plasma protein ratio) rose similarly in both groups, indicating increased barrier permeability. Increased lymphocyte clearance into lung lymph occurred by day 3 but was not associated with lymphocytic sequestration in the lung interstitium. We conclude that pulmonary and systemic microvascular fluid and protein flux exhibit similar changes after bolus or continuous interleukin-2 infusion. These changes are associated with increased clearance of lymphocytes into lung lymph that are not sequestered in the pulmonary interstitium after infusions of shorter duration.

Management of major blood loss utilizing protein-free fluids for volume replacement frequently results in plasma protein depletion and plasma volume expansion. These factors can increase pulmonary transvascular fluid filtration which may lead to life-threatening pulmonary edema. We studied the combined effects of plasma protein depletion and plasma volume expansion on lung lymph flow (QL) in awake sheep prepared with chronic lung lymph fistulae. Animals were first chronically protein-depleted by batch plasmapheresis and then infused for 2 hr with either lactated Ringer's (Hypo/LR; n = 7) or 6% hydroxyethyl starch (Hespan) (Hypo/HES; n = 6). Control normoproteinemic animals (Norm/LR; n = 13) only received lactated Ringer's. Hypoproteinemia alone resulted in an average 2-fold increase in QL over normoproteinemic baseline levels (P less than or equal to 0.05). Infusion of LR into hypoproteinemic animals caused a 7.9-fold increase in QL (P less than or equal to 0.05). By comparison, HES infusion under similar hypoproteinemic conditions limited the increase in QL to 3.2-fold over baseline. We attributed this reduced rise in QL to Hespan's high oncotic pressure, which dramatically widened (by 4-5 mm Hg) the pulmonary-to-lymph oncotic pressure gradient. We did not observe this with LR infusion, or in previous studies employing intravenous infusion of plasma protein. Thus, the oncotic pressure of Hespan appears to significantly limit pulmonary fluid filtration during hypoproteinemia compared to LR. We do not believe that these effects are the results of any changes in microvascular porosity.

To identify lung units associated with liquid leakage into the air space in high-pressure pulmonary edema, we perfused air-inflated dog lung lobes with albumin solution to fill the loose peribronchovascular interstitium. Next, we perfused the lobes for 90 s with fluorescent albumin solution then froze the lobes in liquid nitrogen. This procedure confined the fluorescent perfusate to the liquid flux pathway between the circulation and the air space and eliminated the previously filled peribronchovascular cuffs as a source of the fluorescence that entered the air space. We divided each frozen lobe into three horizontal layers and prepared fluorescence-microscopic sections of each layer. In the most apical layers where alveolar flooding was minimal, 10.6 +/- 21.0% (SD) of alveolar ducts were either fluorescence filled or air filled and continuous with fluorescence-filled alveoli. In the same layers, 11.0 +/- 19.0% of respiratory bronchioles were similarly labeled. No terminal bronchioles in these layers were fluorescence labeled. This suggested that the fluorescent albumin entered the air space across the epithelium of respiratory bronchioles, alveolar ducts, or their associated alveoli. To simulate an alternative explanation, i.e., that fluorescence first entered central airways then flowed into peripheral air spaces, we prepared two additional lobes that we first partially inflated with fluorescent albumin then filled to capacity with air. This pushed the fluorescent solution along the airways into the lung periphery. In these lobes the ciliary lining of bronchi and terminal bronchioles was fluorescence coated. By comparison, cilia in fluorescence-perfused lobes were not coated. We conclude that alveolar flooding in hydrostatic pulmonary edema occurs across the epithelium of alveolar ducts, respiratory bronchioles, or their associated alveoli.(ABSTRACT TRUNCATED AT 250 WORDS)

In the early stages of pulmonary edema, excess liquid leaving the pulmonary exchange vessels accumulates in the peribronchovascular interstitium where it forms large peribronchovascular cuffs. The peribronchovascular interstitium therefore acts as a reservoir to protect the air spaces from alveolar flooding. The rate of liquid accumulation and the liquid storage capacity of the cuffs determine how quickly alveolar flooding is likely to follow once edema formation has begun. To measure the rate and capacity of interstitial filling we inflated 11 isolated degassed dog lung lobes with liquid to an inflation pressure of 14 cmH2O (total lung capacity) for 1-300 min, then froze the lobes in liquid N2. We made photographs of 20 randomly selected 12 X 8-mm cross sections from each lobe and measured cuff volume from the photographs by point-counting. We found that cuff volume increased from 2.2% of air-space volume after 1 min of inflation to 9.3% after 300 min. To measure the driving pressure responsible for cuff formation we used micropipettes to measure subpleural interstitial liquid pressure at the hilum of three additional lobes. With liquid inflation pressure set to 14 cmH2O interstitial pressure rose exponentially to 11.5 cmH2O. Interstitial compliance calculated from our volume and pressure measurements equaled 0.09 ml X cmH2O-1 X g wet wt-1, a value similar to that measured in air-inflated lungs. Goldberg [Am. J. Physiol. 239 (Heart Circ. Physiol. 8): H189-H198, 1980] has likened interstitial filling to the charging of a capacitor, a process that follows a monoexponential time course.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanical properties of the perivascular interstitium surrounding large pulmonary blood vessels are defined in terms of interstitial fluid pressure, interstitial compliance, and interstitial hydraulic resistance. Interstitial pressure is one of the main forces which determine liquid filtration across the microvascular barrier. Interstitial compliance is a measure of the ability of the interstitium to swell with hydration which increases interstitial pressure and reduces the filtration rate. Interstitial pressure and compliance are functions of the elastic properties of the surrounding lung parenchyma and the vessel wall. Solid continuum mechanics are used to describe the behavior of the lung parenchyma. The transport properties of the interstitium are described in terms of a porous material whose fluid resistance is determined by a permeability constant. The dynamics of interstitial fluid are governed by the coupling of the flow with the elastic environment. An electrical analog model is developed to predict the growth of interstitial fluid cuffs during edema formation.

In the initial stages of pulmonary edema, liquid accumulates in the lung interstitium and appears as cuffs around pulmonary vessels. To determine the pattern, rate, and magnitude of cuff formation, we inflated sheep lungs to capacity with liquid (inflation pressure 19 cmH2O) for 3-300 min. After freezing the lobes in liquid N2, we measured perivascular cuff size and total perivascular volume in frozen blocks of each lobe and compared the results with previous measurements in dog lungs. Total cuff volume in sheep lungs reached a maximum value of 5% of air space volume, compared with 9% in dog lungs. In sheep lungs 94% of vessels greater than or equal to 0.5 mm diam and 16% of smaller vessels were surrounded by cuffs. In dog lungs these values were 99 and 47%, respectively. The ratio of cuff area to vessel area reached a maximum of 2.3 in sheep lungs and 3.4 in dog lungs. In an electrical analogue model designed to simulate cuff growth, estimated interstitial resistance to liquid flow was 6-15 times higher than similar estimates in dog lungs. These species differences might be the result of differences in the composition of the interstitial gel or to differences in the mechanical linkage between the lung parenchyma and vessel wall.

We have investigated the mechanism of alveolar liquid filling in pulmonary edema. We excised, degassed, and intrabronchially filled 14 dog lung lobes from nine dogs with 75, 150, 225, or 350 ml of 5% albumin solution, and then air inflated the lobes to a constant airway pressure of 25 cmH2O. By use of micropipettes, we punctured subpleural alveoli to measure alveolar liquid pressure by the servo-null technique. Alveolar liquid pressure was constant in all lobes despite differences in lobe liquid volume and averaged 10.6 +/- 1.3 cmH2O. Thus, in all lobes a constant pressure drop of 14.4 cmH2O existed from airway to alveolar liquid across the air-liquid interface. We attribute this finding, on the basis of the Laplace equation, to an air-liquid interface of constant radius in all the lobes. In fact, we calculated from the Laplace equation an air-liquid interface radius which equalled morphological estimates of alveolar radius. We conclude that in the steady state, alveoli that contained liquid have a constant radius of curvature of the air-liquid interface possibly because they are always completely liquid filled.

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.

In high-pressure pulmonary edema, lung interstitial and air space edema liquids have equal protein concentrations (Am. J. Physiol. 231: 1466, 1976). This suggests that the alveolar-airway barrier separating the air and interstitial spaces is relatively unrestrictive, even without apparent epithelial injury. To estimate the equivalent pore population of the alveolar-airway barrier we inflated each of 18 isolated dog lung lobes for 1 h with a solution of colored tracer of uniform radius. Tracer radii ranged from 1.3 to 405 nm. After freezing the lobes in liquid N2, we measured interstitial tracer concentrations in frozen perivascular cuffs or in samples thawed after dissection from frozen cuffs. Relative to the concentrations instilled, interstitial concentrations ranged from 0.34 for the smallest particles (1.3 and 3.5 nm radius) to zero for particles with radii of 405 nm. From the results we designed a pore model of the alveolar-airway barrier to reproduce the concentrations we measured. No single-pore model could be obtained, although a three-pore model fit the data well. The model results predict that pores with radii of 1, 40, and 400 nm would account for 68, 30, and 2% of total liquid flux, respectively. The majority of liquid flux (68%) would occur through passageways smaller than the smallest tracer we used (1.3 nm radius). We believe the alveolar-airway barrier consists not only of tight intercellular junctions that allow passage of only water and electrolytes but also of a smaller number of large leaks that allow passage of particles up to nearly 400 nm in radius.