Effects of meconium aspiration in isolated perfused rat lungs

Our objective was to study meconium-induced lung injury in isolated perfused rat lungs exposed to anoxia. Our working hypothesis was that meconium-induced lung injury is independent of preexisting hypoxia, and that hypoxia will increase severity of lung injury observed after meconium aspiration. We compared five different groups of animals (n = 5) for pulmonary arterial pressure (PAP), weight lung changes, and TNFalpha expression. Group I had lungs instilled with 4 ml of normal saline. Group II had lungs exposed to 5 min of anoxia. Group III had lungs instilled with 4 ml of 30% filtered human meconium. Group IV had lungs exposed to 5 min of anoxia and then instilled with 4 ml of 30% filtered human meconium. Group V had lungs instilled with 4 ml of 30% unfiltered human meconium. Our subjects were adult Sprague-Dawley rats. The isolated rat lung model was prepared according to Levey and Gast (J Appl Physiol 1966;21:313-316). Lungs were ventilated with room air. Anoxia was caused by the use of N(2). The pulmonary artery was cannulated, and pulmonary arterial pressure and lung weight were measured. Lung weight and pulmonary arterial pressure were monitored for 120 min, and TNFalpha levels were measured in effluent at 15, 30, 60, and 120 min. Experiments were done at the Michael Reese Hospital (Chicago, IL). At the end of the experiment, PAP reached its highest values in group V (10.0 +/- 1.7 mmHg). Final PAPs in groups I-IV were: 4.85 +/- 0.3, 4.99 +/- 0.4, 5.93 +/- 0.3, and 7.25 +/- 0.51 mmHg, respectively). Lung wet weight increased significantly only in groups IV and V vs. group I; at 120 min, they were: 0.96 +/- 0.3 g, P < 0.01, and 1.5 g +/- 0.2 g, P < 0.01, respectively. TNFalpha levels did not change significantly over time in group I. TNFalpha is a marker as well as proprietor of pulmonary inflammatory response. TNFalpha reached its highest levels in groups IV and V: 595 and 753 pg/ml at 120 min, respectively. In conclusion, a short episode of anoxia prior to meconium aspiration may increase lung sensitivity to meconium-induced lung injury. This effect may be moderated by the TNFalpha present in the pulmonary circulation.     

Effect of edema on segmental vascular resistance in isolated perfused rat lungs

We have determined the effect of hydrostatic edema on total and segmental vascular resistances in the rat lung. Lungs of 12 adult rats, body weight 515 +/- 42 g, were isolated and perfused with blood. To investigate the role of vasoactivity on edema effects, we studied two groups of lungs; group I (n = 6) were untreated and group II (n = 6) were treated with papaverine hydrochloride to paralyze the vasculature. Initially blood flow was adjusted to keep pulmonary artery pressure approximately 15 cmH2O, left atrial and airway pressures being 8 and 7 cmH2O, respectively, and then kept unchanged thereafter (18 +/- 3 ml/kg/min). Hydrostatic edema was induced by raising venous pressure and pulmonary artery pressure measured continuously. In 4 lungs from each group, during baseline and after the development of severe edema, we partitioned the pulmonary circulation into arteries, microvessels, and veins by measuring pressures in 20-50 microns diameter subpleural arterioles and venules with the micropipette-Servonull method. Baseline total vascular resistance was similar in the two groups. Interstitial and early alveolar edema did not affect pulmonary vascular pressures. With severe edema (W/D ratio: 17 +/- 2), pressures in pulmonary artery and arterioles increased significantly in both groups; venular pressures did not change. Total resistance increased by 250% in group I and by 224% in group II lungs. Arterial resistance increased 3-5-fold in both groups, as did microvascular resistance. Venous resistances also increased in both groups, although to a lesser extent. The increase in total and segmental vascular resistances was not significantly different in the two groups of lungs. We conclude that in isolated rat lungs only severe edema results in an increase in total vascular resistance, mainly due to an increase in arterial and microvascular resistances, with a smaller increase in venous resistance. This appears to be a mechanical effect of edema on the vasculature and not a result of active vasomotion.     

Effect of surfactant on ventilation-induced mediator release in isolated perfused mouse lungs

The human acute respiratory distress syndrome (ARDS) is a severe pulmonary complication with high mortality rates. To support their vital functions, patients suffering from ARDS are mechanically ventilated. Recently it was shown that low tidal volume ventilation reduces mortality and pro-inflammatory mediator release in these patients, suggesting biotrauma as a side effect of mechanical ventilation. Because the application of exogenous surfactant has been proposed as a treatment for ARDS, we investigated the effect of surfactant on ventilation-induced release of tumor necrosis factor (TNF), interleukin-6 (IL-6) and 6-keto-PGF(1 alpha) (the stable metabolite of prostacyclin) in isolated perfused mouse lungs ventilated with high end-inspiratory pressures. Instillation of 100mg/kg surfactant into the lungs was well tolerated and improved tidal volume, pulmonary compliance and alveolar expansion. Exogenous surfactant increased the ventilation-induced liberation of TNF and IL-6 into the perfusate, but had no effect on the release of 6-keto-PGF(1 alpha). The surfactant preparation used reduced baseline TNF production by murine alveolar macrophages, indicating that the exaggeration of ventilation-induced TNF release cannot be explained by a direct effect of surfactant on these cells. We hypothesize that ventilation-induced mediator release is explained by stretching of lung cells, which is reinforced by surfactant. The findings that in this model of ventilation-induced lung injury exogenous surfactant at the same time improved lung functions and enhanced mediator release suggest that surfactant treatment may prevent barotrauma and augment biotrauma.     

Effect of edema on segmental vascular resistance in isolated perfused rat lungs

We have determined the effect of hydrostatic edema on total and segmental vascular resistances in the rat lung. Lungs of 12 adult rats, body weight 515±42 g, were isolated and perfused with blood. To investigate the role of vasoactivity on edema effects, we studied two groups of lungs; group I (n=6) were untreated and group II (n=6) were treated with papaverine hydrochloride to paralyze the vasculature. Initially blood flow was adjusted to keep pulmonary artery pressure ∼15 cmH₂O, left atrial and airway pressures being 8 and 7 cmH₂O, respectively, and then kept unchanged thereafter (18±3 ml/kg/min). Hydrostatic edema was induced by raising venous pressure and pulmonary artery pressure measured continuously. In 4 lungs from each group, during baseline and after the development of severe edema, we partitioned the pulmonary circulation into arteries, microvessels, and veins by measuring pressures in 20–50μm diameter subpleural arterioles and venules with the micropipette-Servonull method. Baseline total vascular resistance was similar in the two groups. Interstitial and early alveolar edema did not affect pulmonary vascular pressures. With severe edema (W/D ratio: 17±2), pressures in pulmonary artery and arterioles increased significantly in both groups; venular pressures did not change. Total resistance increased by 250% in group I and by 224% in group II lungs. Arterial resistance increased 3–5-fold in both groups, as did microvascular resistance. Venous resistances also increased in both groups, although to a lesser extent. The increase in total and segmental vascular resistances was not significantly different in the two groups of lungs. We conclude that in isolated rat lungs only severe edema results in an increase in total vascular resistance, mainly due to an increase in arterial and microvascular resistances, with a smaller increase in venous resistance. This appears to be a mechanical effect of edema on the vasculature and not a result of active vasomotion.     

Acute effects of leptin on glucose metabolism of in situ rat perfused livers and isolated hepatocytes

OBJECTIVE: To investigate whether leptin interferes directly with glycogenolysis and gluconeogenesis in isolated rat hepatocytes and also in in situ rat perfused livers. ANIMALS: Male albino rats (200-250 g) were used in all experiments. MEASUREMENTS: D-glucose, L-lactate and pyruvate production. RESULTS: In the present study, no differences were found for the rates of glycolysis, as expressed by the areas under the curves, among control (24.2+5.0 mmol?g), leptin (32.0+4.5 mmol?g), glucagon (24.7+3.0 mmol?g), and the leptin + glucagon (23.8+3.4 mmol?g) groups. No difference was found for the rates of glycogenolysis between the control and the leptin perfused livers (15.2+3.9 and 15.0+3.2 mmol?g, respectively). In the presence of glucagon, the areas under the curves for the rate of glycogenolysis rose to 108.6+3.8 mmol?g. When leptin was combined with glucagon, the area under the curve for glycogenolysis was 43. 7+4.3 mmol?g. In fact, leptin caused a reduction of almost 60% (P<0. 001) in the rate of glucagon-stimulated glycogenolysis. Under basal conditions, the addition of leptin (100 ng?ml) to the incubation medium did not elicit any alteration in glucose production by isolated hepatocytes. However, in the presence of leptin, the production of glucose from glycerol (2 mM), L-lactate (2 mM). L-alanine (5 mM) and L-glutamine (5 mM) by the isolated hepatocytes was significantly reduced (30%, 30%, 23% and 25%, respectively). The rate of glucose production (glycogenolysis) by isolated hepatocytes was not different between the control and the leptin incubated groups (445.0+/-91.0 and 428.0+/-72.0 nmol?106 cells?h, respectively). CONCLUSION: We conclude that leptin per se does not directly affect either liver glycolysis or its glucose production, but a physiological leptin concentration is capable of acutely inducing a direct marked reduction on the rate of glucagon-stimulated glucose production in in situ rat perfused liver. Leptin is also capable of reducing glucose production from different gluconeogenic precursors in isolated hepatocytes.     

Acetazolamide reduces hypoxic pulmonary vasoconstriction in isolated perfused rabbit lungs

Carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. We hypothesized that CA inhibition with acetazolamide would inhibit the kinetics and magnitude of hypoxic pulmonary vasoconstriction (HPV). Isolated rabbit lungs were ventilated and perfused in situ at constant flow, with buffer containing red blood cells. Preparations were sequentially challenged with hypoxic (FI(O(2)) 0.05) and/or hypercapnic (FI(CO(2)) 0.10) gas mixtures for 5 or 10 min. In the experimental groups, acetazolamide (33 microM) was added to the perfusate after establishing baseline responses, and gas challenges were repeated; control groups were studied without acetazolamide. Acetazolamide reduced the increase in pulmonary artery pressure (DeltaPAP) and the rate of pressure rise by approximately 30-50% during hypoxia and combined hypoxia/hypercapnia. The reduction in DeltaPAP occurred for both 5 and 10 min challenges. Acetazolamide did not affect expired nitric oxide concentrations. We conclude that acetazolamide reduces both the magnitude and kinetics of HPV by a mechanism that does not involve nitric oxide.     

Eosinophils cause acute edematous injury in isolated perfused rat lungs

Eosinophils produce oxidants and other toxic substances and thus have the potential to cause acute lung injury. We found that addition of normal human eosinophils and the respiratory burst stimulant phorbol myristate acetate to isolated perfused rat lungs acute edematous injury as reflected in weight gain and morphologic changes. Lung to body weight ratio (x 10(3) was 16.7 +/- 3.3 in the experimental group with stimulated eosinophils added compared with 4.7 +/- 0.38 for the control group. Morphologic examination showed both epithelial and endothelial damage. This injury was ameliorated by the addition of catalase, which neutralizes hydrogen peroxide produced during the respiratory burst. Lung/body weight ratio in the group with stimulated eosinophils plus catalase was 7.8 +/- 1.1, and the specimens were indistinguishable from control specimens by histopathologic examination. Our results indicate that eosinophils are capable of causing acute lung injury. This injury is mediated, at least in part, by toxic oxygen products.     

Effects of ligustrazine on the pressure/flow relationship in isolated perfused rat lungs.

Ligustrazine, the synthesized principle of a Chinese herbal remedy shown previously to be a pulmonary vasodilator, was tested in chronically hypoxic and normal rats. Pressure/flow, (P/Q), relations were measured in isolated perfused lungs during normoxia, hypoxia and after reversal of hypoxic vasoconstriction by increasing doses of ligustrazine. P/Q lines were linear over a wide range and extrapolation to the pressure axis gave an intercept which was the effective downstream pressure for flow. In chronically hypoxic rats the slope of the line was steeper and the intercept greater than in control rats, which we attributed to newly muscularized arterioles with tone. Hypoxia caused an increase in slope and intercept in both groups but the intercept increase was greater in chronically hypoxic rats. In both groups of rats increasing doses of ligustrazine given during continued hypoxia caused a fan of lines which moved progressively towards the control normoxic line. In chronically hypoxic rats it required only 2 mg of ligustrazine to bring the line back to the normoxic position, whereas in controls it required 4 mg. In chronically hypoxic rats the change in intercept with every dose was greater than in control rats; this suggests that ligustrazine mainly relaxes the muscle of small collapsible vessels. The action of ligustrazine remained in both control and chronically hypoxic rats after administration of an arginine analogue which blocks synthesis of the endothelial relaxant factor nitric oxide. This and previous evidence suggest that ligustrazine is a non-endothelial-dependent pulmonary vasodilator.     

Effect of dopamine on platelet activating factor induced-pulmonary edema in isolated and perfused rabbit lungs

The effect of dopamine over pulmonary edema induced by PAF was studied. Thirty preparations of rabbit lungs were used: six control preparations (CP), six PAF preparations (PP) in which we injected a dose of 1 microg/kg of rabbit weight and eighteen dopamine preparations (DAP) divided in three groups of six pretreated with a dose of 1-5 (dopaminergic range), 10-20 (Beta range) and 20-30 ug/kg/min (Alpha range) of dopamine, respectively for 30 min, followed by an injection of PAF as in the PP. DAP at Beta and Alpha-adrenergic range decreased pulmonary artery pressure (Pap) as compared to CP, with values of 11.66 (CI 95%: 10.83-12.48), 11.66 (CI 95%: 9.87-13.44) versus 17.12 (CI 95%: 16.12-18.11) cm of water, respectively. DAP in Beta and Alpha-adrenergic range prevented Pap increment as compared to PP, with values of 17.16 (CI 95%: 16.37-17.94), 17.5 (CI 95%: 14.93-20.06) versus 84 cm of water (CI 95%: 71.41-96.58), respectively. Dopamine, at its three ranges inhibited the augmentation of the fluid filtration rate observed in PP with values of 1.01 (CI 95%: 0.77-1.24), 0.03 (CI 95%: 0.01-0.04) and 0.02 g/min (CI 95%: -0.0004-0.03) versus 2.13 g/min (CI 95%: 1.56-2.69), respectively. We concluded that dopamine has a vasodilator effect on Pap and exerts an inhibiting action over PAF effects in pulmonary circulation. Such effects seem to be mainly mediated by Beta-receptors, rather than by dopaminergic receptors.     

Airway responsiveness in isolated perfused rat lungs: effect of thoracic irradiation

We developed techniques for assessing airway reactivity in isolated perfused rat lungs by measuring the lung mechanics changes produced by injection of ACh into the pulmonary circulation. Lung resistance (RL) and dynamic compliance (Cdyn) changed in a dose-response fashion after ACh. We used the preparation to examine the effect of thoracic irradiation on airway responsiveness and pulmonary inflammation. Groups of rats were studied after sham irradiation or 24 h or 72 h after a single dose of 1500 rads. Thoracic irradiation did not alter baseline lung mechanics, but did increase the responsiveness of rat lungs to ACh 72 h after radiation. Radiation was not associated with an increase in neutrophils in lung lavage, airways or peripheral lung tissue. We conclude that thoracic irradiation alters airways reactivity without causing overt pulmonary inflammation, and that isolated perfused lungs can be useful for measurement of airway reactivity.     

Acute cigarette smoke exposure in dogs: the inflammatory response

Acute cigarette smoke causes polymorphonuclear leukocyte (neutrophil, PMN) recruitment to the lung followed by loss of elastase from the recruited cells. Dogs were exposed to cigarette smoke with different oxidant content, bronchoalveolar lavage (BAL) was performed, and the cell distribution in the recovered alveolar lining fluid was analyzed. Exposures were 1, 3, or 6 cigarettes on one or multiple days with a maximum dose of 42 cigarettes. The mean percent PMN present in control lavage was 2.01%, while the mean percent PMN recovered in BAL after a dose of 42 1R1 cigarettes was 13.05%. Recoverable PMN, after a single exposure to three 1R1 cigarettes, also increased from 1.7 to 10.4% by 15 h after cessation of smoke exposure. The cell response for multiple (2 and 7) day exposures was similar. The elastase content per BAL neutrophil decreased relative to peripheral blood PMN from the same animals. No free elastolytic activity was found in BAL, but PMN elastase antigen was present. Increased frequency of cigarette smoke exposure delayed the return to homeostatic cell conditions. The increased PMN accumulation observed may result in an increased proteolytic load in the pulmonary interstitium and contribute to the pathogenesis of emphysema.     

Eicosanoid production in isolated perfused lungs stimulated by calcium ionophore A23187

The patterns of lung eicosanoid production were investigated in five different species by stimulating isolated lungs with calcium ionophore A23187 (10(-5) M). The species studied were the rat, ferret, hamster, guinea pig, and rabbit. The eicosanoids measured included the cyclooxygenase metabolites 6-keto-PGF1 alpha, TxB2, and PGE2 and the lipoxygenase metabolites LTC4, LTD4, and LTB4. Since these metabolites are either retained in the lungs or released into the perfusate, eicosanoids were measured in lung tissue and in the perfusate. In all animal species, calcium ionophore stimulated the production of all the eicosanoid metabolites measured, although in different relative proportions and quantities. The sulfidopeptide LTC4 was predominantly retained in the lung tissue while other eicosanoids were distributed more evenly between tissue and perfusate, with tissue levels generally being higher. The ferret lung produced large quantities of eicosanoids, more than 90% being lipoxygenase products. In contrast, the guinea pig lung produced predominantly cyclooxygenase products, especially TxB2. The rat, hamster, and rabbit lungs showed a more even distribution of cyclooxygenase and lipoxygenase products. Species differences in the ability to produce lipid mediators in the lung may be important in determining the vasoconstrictive, bronchoconstrictive, or inflammatory response to physiologic stimuli.     

Acute cigarette smoke exposure increases alveolar permeability in rabbits

We measured lung clearance of aerosolized technetium-labeled diethylenetriamine pentaacetic acid (99mTcDTPA) as an index of alveolar epithelial permeability in rabbits exposed to cigarette smoke. Eighteen rabbits were randomly assigned to 3 equal-size groups: control, all smoke exposure (ASE), and limited smoke exposure (LSE). Cigarette or sham smoke was delivered by syringe in a series of 5, 10, 20, and 30 tidal volume breaths with a 20-min counting period between each subset of breaths to determine 99mTcDTPA biologic half-life (T1/2). Mean T1/2 minimum (i.e., the smallest T1/2 observed) was significantly lower (p less than 0.05) for ASE and LSE rabbits than by control rabbits. We observed a significant difference at 20 and 30 breath exposures between the control and ASE group mean values (% baseline) for T1/2, arterial blood pressure, and peak airway pressure. A combination of light and electron microscopy showed focal alveolar edema and hemorrhage in the ASE and LSE groups but no alveolar-capillary membrane damage. In summary, acute cigarette smoke exposure increases alveolar permeability as measured by 99mTcDTPA clearance, but there was no detectable ultrastructural alteration of the alveolar-capillary membrane.     

Beneficial effects of hydrocortisone and papaverine on a model of pulmonary embolism induced by autologous blood clots in isolated and perfused rabbit lungs

BACKGROUND AND OBJECTIVE: Mechanical obstruction has been considered the prime determinant of haemodynamic changes after pulmonary embolism (PE); however, the function of vasoconstrictive and inflammatory mediators in the physiopathology of this disease is unclear. The aim of this investigation was to study the effect of an anti-inflammatory and a vasodilator in a setting of induced PE. METHODS: A prospective, laboratory study was undertaken using 30 New Zealand white rabbits. A model of isolated and perfused rabbit lungs was used; PE was induced using autologous blood clots. Six study groups were established (each n = 5): PE without any drug (PG); PE + papaverine (PpG); PE + hydrocortisone (HG); PE in West's Zone III (ZIIIG); PE using acellular perfusate (AG) and PE using acellular perfusate + papaverine (APpG). The pulmonary artery pressure (PAP) and fluid filtration rate (FFR) were continuously measured during the experiments. RESULTS: Increases in PAP and oedema formation were observed in the PG after embolization. The PpG and the APpG showed neither oedema nor significant PAP increases. The HG group developed less oedema and less increase in PAP compared with the PG. The ZIIIG developed oedema the fastest. The AG developed less oedema and increases in PAP compared with the PG. CONCLUSION: These findings suggest that vasoconstriction and inflammatory mediators play an important role in the physiopathology of PE, as neither PAP increases nor oedema were observed in the PpG and a reduction of oedema and PAP was seen in the HG group. The decrease in oedema and PAP in the acellular group strongly suggests a key role of circulating blood cells.     

Calcium-containing particles as a marker of cigarette smoke exposure in autopsy lungs.

It has been recently reported that the major inorganic particle components of cigarette smoke are calcium and potassium carbonates, potassium sulfate and chloride, and quartz. To determine whether any of these particles might serve as a marker of cigarette smoke exposure in human lungs, we used analytical electron microscopy to compare the mineral particle content in two parenchymal sites and two large bronchi from five nonsmoker autopsy lungs and five age- and sex-matched smoker autopsy lungs. Potassium carbonate, sulfate, and chloride were not identified in any lung. The percentage of quartz was the same in both smoker and nonsmoker lungs. However, lungs from smokers contained a large percentage (average 23% of all particles) of particles composed of calcium, carbon, and oxygen (probably calcium carbonate) in all sample sites, whereas lungs from nonsmokers usually contained no such particles or only minute numbers (average 0.1%). We conclude that calcium-containing particles of this type may serve as a marker of cigarette smoke exposure in humans and may prove useful for examining the distribution of smoke particles in the normal and diseased lung.     

Reactive oxygen intermediates reduce inactivation of serotonin in isolated, perfused rat lungs

Reactive oxygen intermediates such as free radicals have been proposed to mediate lung injury. The present work examined whether or not enzymatically generated oxygen metabolites altered serotonin clearance. Isolated, plasma-perfused rat lungs were exposed to xanthine oxidase (XO) and hypoxanthine (HX). Pulmonary arterial pressure (Ppa) and lung weight were recorded. Fulminant edema was defined as a spontaneous weight increase exceeding 500 mg. Inactivation of serotonin was determined by superfusion bioassay. XO and HX reduced serotonin inactivation from 74 +/- 3% (mean +/- SEM) to 62 +/- 2%. This reduction was inhibited by the scavenger enzymes superoxide dismutase (SOD) and catalase and by allopurinol, an inhibitor of XO. Hydrostatic edema and perfusion per se did not decrease the pulmonary clearance of serotonin. XO and HX did not significantly alter Ppa. Fulminant edema developed in four of six lungs after exposure to XO and HX compared with none in the other groups. It was concluded that reactive oxygen intermediates inhibited serotonin inactivation in isolated rat lungs.