Reactive oxygen species-reducing strategies improve pulmonary arterial responses to nitric oxide in piglets with chronic hypoxia-induced pulmonary hypertension.

AIMS: There are no effective treatments for chronic pulmonary hypertension in infants with cardiopulmonary disorders associated with hypoxia, such as those with chronic lung disease. These patients often have poor or inconsistent pulmonary dilator responses to inhaled nitric oxide (iNO) therapy for unknown reasons. One possible explanation for poor responsiveness to iNO is reduced NO bioavailability caused by interactions between reactive oxygen species (ROS) and NO. Our major aim was to determine if strategies to reduce ROS improve dilator responses to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), in resistance pulmonary arteries (PRAs) from a newborn piglet model of chronic pulmonary hypertension. RESULTS: The dilation to SNAP was significantly impaired in PRAs from piglets with chronic hypoxia-induced pulmonary hypertension. ROS scavengers, including cell-permeable and impermeable agents to degrade hydrogen peroxide (H(2)O(2)), improved dilation to SNAP in PRAs from chronically hypoxic piglets. Treatment with agents to inhibit nitric oxide synthase and NADPH oxidase, potential enzymatic sources of ROS, also improved dilation to SNAP in PRAs from hypoxic piglets. INNOVATION: Our studies are the first to utilize a newborn model of chronic pulmonary hypertension to evaluate the impact of a number of potential therapeutic strategies for ROS removal on responses to exogenous NO in the vessels most relevant to the regulation of pulmonary vascular resistance (PRA). CONCLUSIONS: Strategies aimed at reducing ROS merit further evaluation and consideration as therapeutic approaches to improve responses to iNO in infants with chronic pulmonary hypertension.

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to 3 days of hypoxia (Am J Physiol Lung Cell Mol Physiol 295: L881-L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for 3 or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an SOD mimetic (M40403) + polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, SOD2, and catalase were unchanged, whereas expression of SOD1 was reduced, in arteries from piglets raised in hypoxia for 3 or 10 days. Markers of oxidant stress, F(2)-isoprostanes, measured by gas chromatography-mass spectrometry, were increased in PRAs from piglets raised in hypoxia for 3 days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme SOD1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

eNOS and prostanoid enzymes in lungs of newborn piglets with chronic aortopulmonary shunts.

Our purpose was to determine if abundance of proteins underlying nitric oxide (NO) and prostanoid production is altered in lungs of piglets with aortopulmonary shunts. We also evaluated whether shunted piglets exhibit abnormal pulmonary vascular responses to ACh, an endothelium-dependent agent that mediates dilation in part by NO and prostanoid release. At age 4-5 days, piglets underwent either a sham operation or placement of an aortopulmonary shunt. At age 5-6 wk, pulmonary arterial pressure (Ppa) and cardiac output by the thermodilution technique were measured in anesthetized piglets. Ppa responses to the endothelium-dependent agent, ACh, and to a non-endothelium-dependent agent, papaverine, were measured in perfused lungs. An immunoblot technique was applied to homogenates of whole lung tissue and two size groups of pulmonary arteries. In shunted piglets, Ppa and cardiac output were elevated, and Ppa responses to papaverine were reduced. ACh responses were not decreased when expressed relative to Ppa dilation with papaverine. Endothelial nitric oxide synthase (eNOS), cyclooxygenase-1, cyclooxygenase-2, prostacyclin synthase, and thromboxane synthase amounts were unaltered in all lung tissue homogenates. Altered abundance of eNOS and/or prostanoid enzymes does not contribute to the blunted dilation and the elevation in Ppa associated with aortopulmonary shunts in newborn piglets.

L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.

Previously, our laboratory found that pulmonary hypertension developed and lung nitric oxide (NO) production was reduced when piglets were exposed to chronic hypoxia (Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung Cell Mol Physiol 274: L517-L526, 1998). The purposes of this study were to determine whether L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10% O(2) (chronic hypoxia) for 10-12 days. Lung NO production was assessed in isolated lungs from both groups by measuring the perfusate accumulation of nitrites and nitrates (collectively termed NO(-)(x)) before and after addition of L-arginine (10(-2) M) to the perfusate. The rate of perfusate NO(-)(x) accumulation increased by 220% (from 0.8 +/- 0.4 to 2.5 +/- 0.5 nmol/min, P < 0.05) after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 +/- 0. 8 before vs. 3.3 +/- 0.4 nmol/min after L-arginine) in control lungs. In the second series of studies, L-arginine uptake was evaluated by measuring the perfusate concentration of L-[(3)H]arginine at fixed time intervals. The perfusate concentration of L-[(3)H]arginine at each time point was less (P < 0.05) in control than in chronic hypoxic lungs. Thus L-arginine uptake was impaired and may underlie in part the reduction in lung NO production that occurs when piglets are exposed to 10-12 days of chronic hypoxia. Moreover, these findings in isolated lungs lead to the possibility that L-arginine supplementation might increase in vivo lung NO production in piglets with chronic hypoxia-induced pulmonary hypertension.

Nifedipine inhibits pulmonary hypertension but does not prevent decreased lung eNOS in hypoxic newborn pigs.

Therapies to prevent the onset or progression of pulmonary hypertension in newborns have received little study compared with those in adult models. We wanted to determine whether nifedipine treatment prevents the increased pulmonary vascular resistance, blunted pulmonary vascular responses to acetylcholine, and reduced lung endothelial nitric oxide synthase (eNOS) amounts that we have found in a newborn model of chronic hypoxia-induced pulmonary hypertension. Studies were performed with 1- to 3-day-old piglets raised in room air (control) or 10% O2 (hypoxia) for 10-12 days. Some piglets from each group were given nifedipine (3-5 mg/kg sublingually three times a day). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. Pulmonary vascular responses to acetylcholine and eNOS amounts were assessed in excised lungs. The calculated value of the pulmonary vascular resistance for nifedipine-treated hypoxic piglets (0.09 +/- 0.01 cmH(2)O. ml(-1). min. kg) was almost one-half of the value for untreated hypoxic piglets (0.16 +/- 0.01 cmH(2)O. ml(-1). min. kg) and did not differ from the value for untreated control piglets (0.05 +/- 0.01 cmH(2)O. ml(-1). min. kg). Pulmonary arterial pressure responses to acetylcholine and whole lung homogenate eNOS amounts were less for both nifedipine-treated and untreated hypoxic piglets than for untreated control piglets. Nifedipine treatment attenuated pulmonary hypertension in chronically hypoxic newborn piglets despite the persistence of blunted responses to acetylcholine and reduced lung eNOS amounts.

Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs.

To examine the effect of chronic hypoxia on nitric oxide (NO) production and the amount of the endothelial isoform of nitric oxide synthase (eNOS) in lungs of newborn piglets, studies were performed using 1- to 3-day-old piglets raised in room air (control) or 10% O2 (chronic hypoxia) for 10-12 days. Exhaled NO output and plasma nitrites and nitrates (collectively termed NOx-) were measured in anesthetized animals. NOx- concentrations were measured in the perfusate of isolated lungs. eNOS amounts were assessed in whole lung homogenates. In the intact piglets, exhaled NO outputs and plasma NOx- were lower in the chronically hypoxic (exhaled NO output = 0.2 +/- 0.1 nmol/min; plasma NOx- = 10.3 +/- 3.7 nmol/ml) than in control animals (exhaled NO output = 0.8 +/- 0.2 nmol/min; plasma NOx- = 22.3 +/- 4.3 nmol/ml). In perfused lungs, the perfusate accumulation of NOx- was lower in chronic hypoxia (1.0 +/- 0.3 nmol/min) than in control (2.6 +/- 0.6 nmol/min) piglets. The amount of whole lung homogenate eNOS from the chronic hypoxia piglets was 40 +/- 8% less than that from the control piglets. The reduced NO production observed in anesthetized animals or perfused lungs of chronically hypoxic newborn piglets is consistent with the finding of reduced lung eNOS protein amounts. Decreased NO production might contribute to the development of chronic hypoxia-induced pulmonary hypertension in newborns.

Chronic hypoxia alters nitric oxide-dependent pulmonary vascular responses in lungs of newborn pigs.

Almost all of the studies evaluating the effect of chronic hypoxia on lung nitric oxide production have been performed in adult animals. Because results of studies in adult lungs should not be extrapolated to represent the newborn lung, we performed studies to determine whether decreased nitric oxide production might be involved in the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns. We kept newborn pigs in chambers filled with room air (control) or 11-12% O2 for either 3-5 (short) or 10-12 (long) days. Using isolated lungs, we measured pulmonary vascular responses to agents that either stimulate or inhibit the synthesis of nitric oxide. To define the vascular sites of altered production of nitric oxide, we applied the micropuncture technique and measured small venular pressures before and after treatment with a nitric oxide synthesis inhibitor. Pulmonary vascular responses to acetylcholine were blunted in chronically hypoxic piglets of both the short and long groups. The nitric oxide synthesis inhibitor had a different effect in the lungs of control piglets than in those of chronically hypoxic piglets of the long but not of the short group. For the long group, the nitric oxide synthesis inhibitors caused constriction of both arteries and veins in lungs of control but not of chronically hypoxic piglets. These findings support the idea that decreased pulmonary vascular nitric oxide production occurs with chronic hypoxia in newborn pigs and might therefore contribute to the pathogenesis of pulmonary hypertension in newborns.

Effect of chronic hypoxia on pulmonary vascular pressures in isolated lungs of newborn pigs.

Our purposes were to determine whether chronic alveolar hypoxia altered pulmonary vascular pressures in lungs of newborn pigs, evaluate the contribution of smooth muscle tone to alterations in pulmonary vascular pressures, and examine whether chronic hypoxia altered pulmonary vascular reactivity to acute hypoxia. We kept 24- to 72-h-old pigs in chambers filled with room air (control) or 11-12% O2 (chronic hypoxia) for either 3-5 (short) or 10-12 (long) days. We used isolated lungs and applied micropuncture and vascular occlusion techniques to measure pressure in 10- to 30-microns-diam venules and inflow occlusion and outflow occlusion pressures before and after the addition of the smooth muscle dilator papaverine or before and after inflation of the lungs with a hypoxic gas mixture. For pigs in both the short and long groups, pulmonary arterial pressure was the only vascular pressure that was greater in chronically hypoxic than in control lungs. Increased smooth muscle tone was the primary source of the change in pulmonary arterial pressure with short hypoxia, whereas morphometric changes contributed to the change in pulmonary arterial pressure with long hypoxia. Exposure of newborn pigs to different lengths of alveolar hypoxia is a useful model to study postnatal pulmonary hypertension in newborns and infants.

Developmental differences in vascular responses to hypoxia in lungs of rabbits.

Our purpose was to determine whether postnatal age and prostaglandins influence the sites of hypoxic vasoconstriction in lungs of rabbits. To do this, we used the micropuncture technique to measure pressures in 20- to 80-microns-diam subpleural arterioles and venules during sequential inflation of lungs of newborn and adult rabbits with normoxic (21% O2-7-10% CO2-69-72% N2) and hypoxic (90-93% N2-7-10% CO2) gas mixtures. Indomethacin (40 micrograms/ml) was added to the perfusate of some lungs of each age group. During hypoxia in untreated lungs of newborn rabbits, both pulmonary arterial and 20- to 80-microns-diam arteriolar pressure increased by 5%, whereas 20- to 80-microns-diam venular pressure remained the same. In contrast, during hypoxia in untreated lungs of adult rabbits, pulmonary arterial pressure increased by 48%, whereas 20- to 80-microns-diam arteriolar pressure decreased slightly and 20- to 80-microns-diam venular pressure did not change. Regardless of the presence of indomethacin, location of vessels used for micropuncture, or level of left atrial pressure, pulmonary arterial pressure was the only measured vascular pressure that increased with hypoxia in adult lungs. Thus, in adult lungs, the site of hypoxia-induced vasoconstriction was limited to arteries > 80 microns diam, whereas in newborn lungs the site of hypoxia-induced vasoconstriction included vessels both larger and smaller than 20- to 80-microns-diam arteries. This age-related difference in the sites of hypoxia-induced vasoconstriction was not found in indomethacin-treated lungs.

Micropipette and vascular occlusion pressures in isolated lungs of newborn lambs.

We compared measurements made by vascular occlusion, double occlusion, and micropuncture techniques in isolated perfused lungs of 3- to 10-day-old lambs. After adding indomethacin to the perfusate of 16 lungs, we used the micropuncture technique to measure pressures in 20- to 80-microns-diam arterioles (Pa80), 20- to 50-microns-diam venules (Pv20), and 50- to 150-microns-diam venules (Pv150). The vascular occlusion and double occlusion techniques were used in these same lungs to measure inflow occlusion (Pao), outflow occlusion (Pvo), and double occlusion pressures (Pdo). In 14 other lungs without indomethacin added to the perfusate, we measured Pv20 and Pvo before and during alveolar hypoxia and/or before and after the addition of papaverine to the perfusate. In indomethacin-treated lungs, Pao was greater than Pa80, Pvo equaled Pv20 and Pv150, and Pdo was the same as Pa80, Pv20, and Pv150. In lungs without indomethacin treatment, Pvo was less than Pv20 both before and during hypoxia but Pvo equaled Pv20 after papaverine was added to the perfusate. Thus, in indomethacin-treated lungs of newborn lambs, Pao reflected pressure in arteries > 80 microns diam, Pdo reflected pressure between 80-microns-diam arteries and 150-microns-diam veins, and Pvo was similar to pressure in veins both as small as 20 microns diam and as large as 150 microns diam. However, without indomethacin there was a gradient between Pvo and Pv20 both before and during hypoxia but not after vasomotor tone was reduced with papaverine. Thus, the correlation between Pvo and micropuncture pressure in Pv20 and Pv150 was not constant and appeared related to vasomotor tone.

Effect of airway and left atrial pressures on microvascular and interstitial pressures in adult lungs.

The purpose of this study was to determine the effect of lung inflation and left atrial pressure on the hydrostatic pressure gradient for fluid flux across 20- to 80-microns-diam arterioles and venules in isolated perfused lungs of adult rabbits. We used the micropuncture technique and measured microvascular or interstitial pressures at constant airway pressures of 5 and 15 cmH2O with left atrial pressure adjusted above (zone 3 conditions) or below (zone 2 conditions) airway pressure. Only in lungs inflated to the higher airway pressure did a reduction in left atrial pressure below airway pressure result in concomitant reductions in venular pressure. This suggests that the site of flow limitation in zone 2 shifted from venules > 80 microns diam toward vessels <20 microns diam with inflation from 5 to 15 cmH2O. With the lungs under zone 3 conditions, both transarteriolar and transvenular gradients (microvascular-interstitial pressures) were greater at the higher compared with the lower airway pressure. In contrast, transarteriolar and transvenular gradients changed in opposite directions when compared at the two inflation pressures under zone 2 conditions. Counteracting changes in transmicrovascular gradients make it difficult to predict the effect on fluid filtration from lung inflation under zone 2 conditions. When zone 3 conditions are maintained during inflation, the tendency for edema formation should increase.

Pulmonary venous pressure increases during alveolar hypoxia in isolated lungs of newborn pigs.

The purpose of this study was to determine whether pulmonary venous pressure increases during alveolar hypoxia in lungs of newborn pigs. We isolated and perfused with blood the lungs from seven newborn pigs, 6-7 days old. We maintained blood flow constant at 50 ml.min-1.kg-1 and continuously monitored pulmonary arterial and left atrial pressures. Using the micropuncture technique, we measured pressures in 10 to 60-microns-diam venules during inflation with normoxic (21% O2-69-74% N2-5-10% CO2) and hypoxic (90-95% N2-5-10% CO2) gas mixtures. PO2 was 142 +/- 21 Torr during normoxia and 20 +/- 4 Torr during hypoxia. During micropuncture we inflated the lungs to a constant airway pressure of 5 cmH2O and kept left atrial pressure greater than airway pressure (zone 3). During hypoxia, pulmonary arterial pressure increased by 69 +/- 24% and pressure in small venules increased by 40 +/- 23%. These results are similar to those obtained with newborn lambs and ferrets but differ from results with newborn rabbits. The site of hypoxic vasoconstriction in newborn lungs is species dependent.

Effects of lung inflation and blood flow on capillary transit time in isolated rabbit lungs.

In a previous study, direct measurements of pulmonary capillary transit time by fluorescence video microscopy in anesthetized rabbits showed that chest inflation increased capillary transit time and decreased cardiac output. In isolated perfused rabbit lungs we measured the effect of lung volume, left atrial pressure (Pla), and blood flow on capillary transit time. At constant blood flow and constant transpulmonary pressure, a bolus of fluorescent dye was injected into the pulmonary artery and the passage of the dye through the subpleural microcirculation was recorded via the video microscope on videotape. During playback of the video signals, the light emitted from an arteriole and adjacent venule was measured using a video photoanalyzer. Capillary transit time was the difference between the mean time values of the arteriolar and venular dye dilution curves. We measured capillary transit time in three groups of lungs. In group 1, with airway pressure (Paw) at 5 cmH2O, transit time was measured at blood flow of approximately 80, approximately 40, and approximately 20 ml.min-1.kg-1. At each blood flow level, Pla was varied from 0 (Pla less than Paw, zone 2) to 11 cmH2O (Pla greater than Paw, zone 3). In group 2, at constant Paw of 15 cmH2O, Pla was varied from 0 (zone 2) to 22 cmH2O (zone 3) at the same three blood flow levels. In group 3, at each of the three blood flow levels, Paw was varied from 5 to 15 cmH2O while Pla was maintained at 0 cmH2O (zone 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Longitudinal distribution of pulmonary vascular pressures as a function of postnatal age in rabbits.

The major purpose of this study was to determine whether the longitudinal distribution of pulmonary vascular pressures changes with postnatal age in rabbits. Using the direct micropuncture technique, we measured pressures in 20- to 80-microns-diam arterioles and venules in isolated lungs of rabbits of different postnatal ages. To determine the contribution of vasomotor tone, we added the vasodilator papaverine to the perfusate of some lungs of each age group. We compared vascular pressures measured at blood flow rates chosen to approximate in vivo cardiac outputs. In untreated lungs, the resistance across 20- to 80-microns-diam microvessels decreased from 12- to 72-h-old (0.022 +/- 0.009 cmH2O.min.kg.ml-1) to 5- to 15-day-old rabbits (0.008 +/- 0.007 cmH2O.min.kg.ml-1) and remained at this lower level in adults (0.013 +/- 0.008 cmH2O.min.kg.ml-1). In contrast, in papaverine-treated lungs, the resistance across 20- to 80-microns-diam microvessels did not change between 12- to 72-h-old (0.007 +/- 0.005 cmH2O.min.kg.ml-1) and 5- to 15-day-old rabbits (0.005 +/- 0.002 cmH2O.min.kg.ml-1) but increased between 5- to 15-day-old and adult rabbits (0.014 +/- 0.007 cmH2O.min.kg.ml-1). Thus vasomotor tone contributed to the postnatal change in the distribution of vascular pressures across lungs of rabbits.

Effect of blood flow rate and blood flow history on newborn pulmonary microcirculation.

The purpose of this study was to determine whether increased pulmonary blood flow and/or the history of pulmonary blood flow alters microvascular pressures in lungs of newborns. Using the direct micropuncture technique, we measured pressures in 20- to 60-microns-diameter arterioles and venules in isolated lungs of newborn rabbits at consecutive blood flow rates of 50 (baseline), 100, and/or 200 ml.min-1.kg-1. Then in some lungs we returned blood flow rate to baseline and repeated microvascular pressure measurements. We kept left atrial pressure the same at all blood flow rates. When blood flow rate increased and left atrial pressure was maintained constant, pulmonary arterial, 20- to 60-microns-diameter arteriolar, and 20- to 60-microns-diameter venular pressures increased such that the percentage of total pressure drop that occurred across veins increased. When we returned blood flow to baseline, venular pressure returned to baseline, but arteriolar and pulmonary arterial pressures returned to values less than baseline so that the percentage of the total pressure drop that occurred across microvessels decreased. Thus both blood flow rate and blood flow history are important determinants of the longitudinal distribution of pulmonary vascular pressures across newborn lungs. These findings also suggest that in newborn lungs venules greater than 60 microns diameter are poorly distensible such that higher blood flow rates result in increased microvascular pressures. Hence, under conditions of increased pulmonary blood flow, such as occurs with left to right shunts, the tendency for edema formation will increase in newborn lungs even if left atrial pressure does not increase.

Regional variations in lung expansion in rabbits: prone vs. supine positions.

We studied the vertical gradient in lung expansion in rabbits in the prone and supine body positions. Postmortem, we used videomicroscopy to measure the size of surface alveoli through transparent parietal pleural windows at dependent and nondependent sites separated in height by 2-3 cm at functional residual capacity (FRC). We compared the alveolar size measured in situ with that measured in the isolated lungs at different deflationary transpulmonary pressures to obtain transpulmonary pressure (pleural surface pressure) in situ. The vertical gradient in transpulmonary pressure averaged 0.48 +/- 0.16 (SD) cmH2O/cm height (n = 10) in the supine position and 0.022 +/- 0.014 (SD) cmH2O/cm (n = 5) in the prone position. In mechanically ventilated rabbits, we used the rib capsule technique to measure pleural liquid pressure at different heights of the chest in prone and supine positions. At FRC, the vertical gradient in pleural liquid pressure averaged 0.63 cmH2O/cm in the supine position and 0.091 cmH2O/cm in the prone position. The vertical gradients in pleural liquid pressure were all less than the hydrostatic value (1 cmH2O/cm), which indicates that pleural liquid is not generally in hydrostatic equilibrium. Both pleural surface pressure and pleural liquid pressure measurements show a greater vertical gradient in the supine than in the prone position. This suggests a close relationship between pleural surface pressure and pleural liquid pressure. Previous results in the dog and pony showed relatively high vertical gradients in the supine position and relatively small gradients in the prone position. This behavior is similar to the present results in rabbits. Thus the vertical gradient is independent of animal size and might be related to chest shape and weight of heart and abdominal contents.

Pleural protein concentration and liquid volume in spontaneously hypertensive rats.

To determine the effect of systemic vascular hypertension on fluid balance in the pleural space, we studied the spontaneously hypertensive rat (SHR) and its genetic normotensive control, the Wistar-Kyoto rat (WKY). We measured arterial and venous pressures, total protein and albumin concentrations of pleural liquid and plasma, pleural space thickness, and pleural surface pressure in SHR and WKY that were matched for weight (260-300 g). Protein concentration was measured by a manual Biuret test and albumin concentration was measured by the bromcresol green colorimetric method. Pleural liquid thickness was measured in situ using light microscopy. Pleural surface pressure was assumed to equal pleural liquid pressure. In the SHR, total protein and albumin concentrations in pleural liquid were lower than in WKY, and pleural space thickness was larger in SHR than in WKY. These results are consistent with a higher capillary pressure and greater fluid filtration in SHR.

Pleural space thickness in situ by light microscopy in five mammalian species.

The thickness of the pleural space was measured by a focusing method using a light microscope (X157, 2.5-micron depth of focus). In anesthetized animals, thin transparent parietal pleural windows were made by dissection of intercostal muscle. Multiple postmortem measurements were made of the combined thickness of the pleural space and the window by focusing in sequence on the lung surface and on 1- to 2-micron tantulum particles sprayed on the window. The window thickness was measured after creating a pneumothorax and retracting the lungs. In supine rabbits the pleural space measured at various heights on the costal surface was of uniform thickness (16 micron) except for a thicker region (62 micron) located within 3 mm of the most dependent part of the lung. The thicker region reverted to the uniform thickness after it was placed in a nondependent position by inverting the animal from the supine to prone position, indicating fluid drainage by gravity. In the prone position near midchest, pleural space thickness (t) averaged 6.9 micron in the mouse, 10.2 in the rat, 17.2 in the rabbit, 18.3 in the cat, and 23.6 in the dog. Animals of larger body mass (M, kg) had a wider pleural space: t = 13.1 X M0.20. There was no contact between the two pleurae, indicating that fluid lubrication facilitates sliding between the lung and chest wall. Based on the t vs. M relationship and estimates of the viscous flow of pleural liquid, pleural fluid exchange rate would be proportional to body mass and the work of sliding as a fraction of the work of breathing would be smaller in larger animals.

Alveolar liquid pressure measured using micropipets in isolated rabbit lungs.

The relationships among alveolar liquid pressure (Pliq), transpulmonary pressure (Ptp), and alveolar edema were studied in isolated rabbit lungs. Different amounts of edema were induced by instilling measured amounts of normal saline via the trachea. Micropipets were used in conjunction with a servonulling pressure measuring system to measure Pliq. Pliq was -1 cm H2O relative to alveolar air pressure (Palv) at Ptp of 3 cm H2O and decreased to -16 cm H2O at Ptp of 25 cm H2O. Pliq increased slightly as wet-to-dry weight ratio (W/D) of the lungs increased from 5.8 to 16. Alveolar surface tension estimated from these values of Pliq - Palv using an alveolar radius of curvature of 40 micron at Ptp of 25 cm H2O was consistent with the direct measurements of Schürch. In lungs made edematous by treating with oleic acid, values of Pliq were not different from those measured in lungs made edematous by normal saline instillation (control). Pressure-volume behaviour of the oleic acid treated lungs showed a marked reduction in lung volume at each Ptp value compared to the behavior in the control lungs. Most of this reduction in lung volume was removed by washing the airways with the bronchodilator, isoproterenol. We conclude that alveolar surface tension was not increased in the oleic acid treated lungs. The apparent increase in lung static recoil was most likely caused by the constriction of small airways.