Leakage of macromolecules in ventilated and unventilated segments of preterm lamb lungs.

The movement of macromolecules into and out of unventilated lung segments was evaluated in prematurely delivered and ventilated lambs. Seven lambs at 130 days gestational age had a bronchial balloon placed at birth before the first breath to obstruct the left lower lobe. Surfactant and 131I-albumin were instilled into the left lower lobe while surfactant and 125I-albumin were instilled into the remaining lung, and 70,000 molecular weight [3H]dextran was given into the vascular space at birth. Twenty-five percent of the lung by weight was not ventilated, and 24% of the total leak of dextran from the vascular space was recovered in the unventilated lungs at 3 h. An epithelial leak of protein from the two lung regions was documented by the loss of 11.4 and 18.4% of the labeled albumins in the nonventilated and ventilated lung regions, the appearance of 4.9 and 7.5% of the airway-instilled albumin in the vascular space from the nonventilated and ventilated lung regions, and the recovery of the labeled albumins in the carcasses of the lambs. The bidirectional flux of macromolecules was larger in the ventilated than in the nonventilated lung regions, indicating that ventilation can increase the leak of protein in the preterm lung. The lung areas that were never exposed to ventilation or oxygen also demonstrated a large bidirectional flux of macromolecules, a finding not present in the fetus, fullterm newborn, or adult. These findings indicate that ventilation is not solely responsible for the increased protein leak found in preterm lungs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

Surfactant metabolism in surfactant-treated preterm ventilated lambs

Preterm lambs were delivered at 132 days gestational age, treated with 100 mg/kg radiolabeled natural sheep surfactant or Surfactant TA, and ventilated for times up to 24 h. Compared with an untreated group that developed respiratory failure by 5 h, both surfactant-treated groups had stable respiratory function to 24 h. Although only approximately 13% of the labeled surfactant phosphatidylcholine was recovered by alveolar wash at 24 h, there was no significant loss of the labeled phosphatidylcholine from the lungs. Labeled palmitic acid intravascularly injected at 1 h of age comparably labeled lung phosphatidylcholine in the three groups of lambs at 5 h; however, only approximately 0.5% of the labeled phosphatidylcholine was secreted to the air spaces of surfactant-treated lambs at 24 h. Labeled lysophosphatidylcholine given with the natural sheep surfactant was taken up by the lungs, converted to phosphatidylcholine with 30-40% efficiency, and resecreted to the air spaces, demonstrating recycling of a phospholipid. The large surfactant aggregates recovered from alveolar washes by centrifugation were surface active and contained approximately 76% of the air-space phosphatidylcholine in both surfactant-treated groups. Although clinical status was comparable, alveolar washes and surfactant subfractions from Surfactant TA-treated lambs had better surface properties than did sheep surfactant-treated lambs. These studies identified no detrimental effects of surfactant treatments on endogenous surfactant metabolism and indicated that the surfactants used for treatments were recycled by the preterm ventilated lamb lung.     

Carbon dioxide concentrations in unventilated IVC cages

The use of individually ventilated cage (IVC) systems has become more common worldwide. The various systems are becoming more and more sealed in order to protect the animals against infections and the staff against allergens; which, however, may lead to problematic CO2 concentrations, if the cages are left unventilated. In this study it is shown that, depending on how tight the cage is and the number of animals housed in each cage, CO2 inside the cage within 2 h will increase to levels of between 2 and 8%.     

Glycerolipid metabolism during early amphibian embryogenesis. Neutral lipid involvement in fertilization triggered events

In order to study glycerolipid metabolism during early embryonic development, Bufo arenarum Hensel female toads were administered [1-3-(14)C]glycerol together with hormonal stimulus. Triglycerides comprised 60% of the total label in all developmental stages analyzed. The highest specific activity was observed in phosphatidic acid which underwent a net decrease as a function of development. Phosphatidylcholine was the most actively synthesized phosphoglyceride. A significant decrease in the total lipid labeling, mainly accounted for by triglycerides, was detected at fertilization time concomitant with a net diminution in the content of these neutral lipids. Subcellular studies showed that yolk platelets contain about 99% of the total oocyte triglycerides suggesting that massive breakdown would be confined to this fraction. Present findings evidence that the de novo lipid biosynthesis is operating during the developmental analyzed period. In addition, they would suggest the active role of triglycerides in the energetic events taking place around fertilization.     

Cholesterol metabolism in fibroblasts from rabbits resistant to diet-induced hypercholesterolemia

We have previously described a colony of New Zealand White rabbits that are resistant to hypercholesterolemia when fed a cholesterol-enriched diet. The present studies used skin fibroblasts obtained from normal and hypercholesterolemia-resistant rabbits to investigate cholesterol metabolism and lipid composition in vitro. The lipid compositions of the two cell lines after incubation in either fetal calf serum or lipoprotein-deficient serum were similar. The conversion of radiolabeled acetate into sterol and phospholipids was higher in resistant fibroblasts than in normal fibroblasts. In contrast, incorporation of radiolabeled oleic acid into cholesteryl ester was significantly lower in resistant fibroblasts than in normal cells. In parallel experiments, the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was higher and acyl-coenzyme A:cholesterol acyltransferase activity was lower in resistant cells compared to normal cells. Furthermore, binding, uptake, and degradation of normal rabbit 125I-labeled LDL (low density lipoproteins) were 30% higher in resistant than in normal fibroblasts. These observations are consistent with results from previous studies of cholesterol metabolism in the liver membranes of these rabbits. The results indicate that extrahepatic cells (such as fibroblasts) from the resistant rabbit exhibit the same altered cholesterol metabolism as that found in the hepatic tissues of these rabbits. These studies suggest that the resistant rabbit may provide an in vivo and in vitro system for studying the mechanisms by which some individuals of a species can minimize the effect of dietary cholesterol on the development of hypercholesterolemia and atherosclerosis.     

Effect of tidal volume and frequency on airway responsiveness in mechanically ventilated rabbits

Shen, X., S. J. Gunst, and R. S. Tepper. Effect oftidal volume and frequency on airway responsiveness in mechanically ventilated rabbits. J. Appl. Physiol.83(4): 1202-1208, 1997.We evaluated the effects of the rate andvolume of tidal ventilation on airway resistance (Raw) duringintravenous methacholine (MCh) challenge in mechanically ventilatedrabbits. Five rabbits were challenged at tidal volumes of 5, 10, and 20 ml/kg at a frequency of 15 breaths/min and also under static conditions(0 ml/kg tidal volume). Four rabbits were subjected to MCh challenge atfrequencies of 6 and 30 breaths/min with a tidal volume of 10 ml/kg andalso under static conditions. In both groups, the increase in Raw with MCh challenge was significantly greater under static conditions thanduring tidal ventilation at any frequency or volume. Increases in thevolume or frequency of tidal ventilation resulted in significant decreases in Raw in response to MCh. We conclude that tidal breathing suppresses airway responsiveness in rabbits in vivo. The suppression ofnarrowing in response to MCh increases as the magnitude of the volumeor the frequency of the tidal oscillations is increased. Our findingssuggest that the effect of lung volume changes on airway responsivenessin vivo is primarily related to the stretch of airway smooth muscle.

     

Effect of granulocyte depletion in a ventilated surfactant-depleted lung

In a previous paper Cutz, Bryan et al. showed that in rabbits after repetitive lung lavage high-frequency oscillatory ventilation maintained excellent gas exchange and did not cause hyaline membrane formation (J. Appl. Physiol. 55: 131-138, 1983). In contrast, conventional mechanical ventilation had poor gas exchange and extensive hyaline membrane formation and we attributed these differences to mechanical barotrauma. However, we completely overlooked the large number of granulocytes in the damaged lung. To investigate this using the same model we have used mechanical ventilation on two groups of rabbits, one with normal granulocytes, the other depleted of granulocytes by pretreatment with nitrogen mustard. The nondepleted rabbits had poor gas exchange, a substantial protein leak into the lung and extensive hyaline membranes. The depleted animals had good gas exchange, a very small protein leak and no hyaline membranes. Repletion of granulocytes from donor rabbits lead to poor gas exchange and hyaline membrane formation. It is concluded that lung lavage causes prompt margination of granulocytes which become activated by the ongoing epithelial barotrauma of conventional ventilation.     

Lung aeration and lung water dynamics in artificially ventilated newborn lambs

To the present lung liquid dynamics in the immediate neonatal period have been measured mainly by gravimetric techniques. This paper explores lung aeration and lung water dynamics in seven fetal lambs between 139 and 142 days of gestation delivered by caesarean section and ventilated on a constant-volume respirator. After caesarean section and instrumentation, fetal lambs were quickly transferred to a warm X-ray table and connected to a volume-cycled respirator. X-ray fluoroscopic images of the chest commenced before the first breath and were recorded on video tape. After 1 h of ventilation, measurements were made of pulmonary blood volume, and lung samples were taken for wet weight and dry weight analysis. Fluoroscopic image brightness was calibrated by comparison with images obtained from a water wedge, which extended across the X-ray field. Thus image brightness was related to "equivalent water path length" through the thorax. Within a defined lung field, image brightness increases as the amount of lung water in the path of the X-ray beam is reduced. This occurs rapidly at first and then more slowly over the remaining hour. There was considerable variation between the reduction of liquid in the lung fields examined within the one animal, as well as the absolute amount of fluid that had been cleared during the 1st h.     

The metabolism of estriol in rabbits

Rabbits have been shown to excrete 6, 7-³H-estriol, its conjugates and metabolites preponderantly in the bile during the initial 4 hours following the I.V. injection of the labeled steroid. The amount of radioactivity excreted in the urine was $\text{1}\text{3}$ of that in the bile. Since in intact rabbits most of the injected radioactivity of ³H-estriol is excreted in the urine over a period of days (and very little in the feces), it appears that estriol and its conjugates and metabolites are involved in an efficient enterohepatic. circulation. In the bile, the preponderant metabolite of ³H-estriol was the 3-glucosiduronate. Even though the latter constituted a substantial part of the urinary metabolites, other conjugates and metabolites of estriol were present in considerable amounts. It is possible that the latter have resulted from gastro-intestinal and/or renal metabolism. Incubation of rabbit liver with estriol led to 75% conjugation with glucuronic acid in the 3-position.     

Fuel metabolism in fasted newborn rabbits

Newborn rabbits delivered by Caesarean section at term were fasted for 72 h at 36 degrees C. Despite the abrupt interruption of maternal supply of energy substrates, glycaemia remains stable for 4 h after birth. This can be related to glucose production via rapid liver glycogenolysis; however, indirect evidence suggests that gluconeogenesis could also contribute to glucose production during this period. There is a selective decrease in the concentrations of gluconeogenic substrates and a suitable hormonal environment for gluconeogenesis as decreased insulin and increased glucagon concentration just after birth. The relative hypoglycaemia which develops after 6 h of life (2.6 mM at 72 h), despite high blood concentrations of non-esterified fatty acids and ketone bodies is not due to a deficient gluconeogenesis per se, as injection of gluconeogenic substrates to 72 h fasted newborns produces a three-fold increase in plasma glucose concentration. It is suggested that this relative hypoglycaemia is secondary to limited gluconeogenic substrate availability in the form of low circulting concentrations of gluconeogenic amino acids.     

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.     

Monokine-induced acute lung injury in rabbits

Interleukin-1 (IL-1) mediates components of the acute phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied in unanesthetized rabbits the effects of intravenous divided dose infusions of a murine monokine preparation containing IL-1 activity, on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability. Monokine administration induced significant (P less than 0.01) granulocytopenia as well as a significant (P less than 0.001) increase in mean alveolar septal wall granulocytes per high power field (HPF) compared with saline-injected controls. Infusions of the monokine preparation significantly (P less than 0.005) increased lung wet-to-dry weight ratios as well as significantly (P less than 0.025) increased pulmonary extravasation of radiolabeled albumin. Electron microscopic analysis of lung sections obtained from monokine-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural tracer. We conclude that a monokine preparation containing IL-1 activity can induce profound granulocytopenia, pulmonary leukostasis, and acute pulmonary vascular endothelial injury.     

Alveolar and lung liquid clearance in anesthetized rabbits

Alveolar and lung liquid clearance were studied over 8 h in intact anesthetized ventilated rabbits by instillation of either isosmolar Ringer lactate (2 ml/kg) or autologous plasma (2 or 3 ml/kg) into one lower lobe. The half time for lung liquid clearance of the isosmolar Ringer lactate was 3.3 h and that for plasma clearance was 6 h. In the plasma experiments, the alveolar protein concentration after 1 h was 5.2 +/- 0.8 g/dl, which was significantly greater than the initial instilled protein concentration of 4.3 +/- 0.7 g/dl (P less than 0.05). Thus alveolar protein concentration increased by 21 +/- 12% over 1 h, which matched clearance from the entire lung of 19 +/- 11% of the instilled volume. Overall the rate of alveolar and lung liquid clearance in rabbits was significantly faster than in prior studies in dogs and sheep. The fast alveolar liquid clearance rate in rabbits was not due to higher endogenous catecholamine release, because intravenous and alveolar (5 x 10(-5) M) propranolol did not slow the clearance. Also, beta-adrenergic therapy with alveolar terbutaline (10(-5) or 10(-4) M) did not increase the alveolar or lung liquid clearance rates. Phloridzin (10(-3) M) did not slow alveolar liquid clearance. However, amiloride (10(-4) M) inhibited 75% of the basal alveolar liquid clearance in rabbits, thus providing evidence that alveolar liquid clearance in rabbits depends primarily on sodium-dependent transport. This rabbit study provides further evidence for important species differences in the basal rates of alveolar liquid and solute clearance as well as the response to beta-adrenergic agonists and ion transport inhibitors.     

Biphasic response to lipopolysaccharide from E. coli in the isolated ventilated blood-perfused rat lung.

We characterised early circulatory and respiratory responses to lipopolysaccharide from E. coli (LPS, serotype 0127:B8) in the isolated, ventilated and perfused rat lung preparation. Lungs were isolated from anaesthetised Wistar rats and perfused with full blood, platelet rich plasma (PRP), platelet poor plasma (PPP) or Krebs-Henseleit solution (KH). LPS (300 microg/ml) injected into the blood-perfused lung induced a characteristic biphasic response consisting of an immediate, transient decrease in respiratory tidal volume and an increase in pulmonary perfusion pressures followed by a delayed decrease in respiratory tidal volume. An immediate respiratory/circulatory response to LPS was of considerable magnitude only in full blood-perfused lung whereas the delayed response was fully expressed irrespective whether blood, PRP, PPP or KH was used for the lung perfusion. Immediate respiratory/circulatory response was inhibited by WEB 2170 (100 microM), a PAF receptor antagonist, and by camonagrel (300 microM), a TXA2 synthase inhibitor, but not by MK 571 (100 microM), a cysteinyl leukotriene receptor antagonist. Delayed respiratory response was inhibited by camonagrel only. In summary, we demonstrated that the immediate coupled respiratory/circulatory response is mediated by blood cell-derived PAF and TXA2 whereas the delayed uncoupled respiratory response is mediated by lung parenchyma-derived TXA2.