The effect of hexafluorocyclobutene on rat bronchoalveolar lavage fluid surfactant phospholipids and alveolar type II cells

Hexafluorocyclobutene (HFCB), a reactive organohalogen gas, causes overwhelming pulmonary oedema. We investigated its effect on the rat lung surfactant system, comparing its action on type II pneumocytes with air-exposed rats. The inflammatory cell population and protein content of bronchoalveolar lavage fluid was analysed following exposure to air or HFCB (LCt30). Six rat lung phospholipids were measured by high-performance liquid chromatography, following solid phase extraction (SPE) from lavage fluid. Transmission electron microscopy (TEM) was used to visualise effects on alveolar type II cell ultrastructure. HFCB caused changes in cell populations and increased lavage fluid protein compared to controls, suggesting a permeability oedema. Changes in the total amount and percentage composition (sustained decrease in phosphatidylglycerol and phosphatidylcholine) of surfactant phospholipids also occurred. TEM observations indicated no direct ultrastructural damage to the type II cells, but showed initial, rapid release of surfactant into the alveolar space. HFCB altered the surfactant system in a manner similar to that shown following another reactive organohalogen gas, perfluoroisobutene (PFIB), but differently to that after phosgene. These differences suggest different mechanisms of action even though pulmonary oedema is the final injury for all gases. Better knowledge of the mechanisms involved will improve prospects for prophylactic/therapeutic intervention.     

Enhanced pulmonary delivery of insulin by lung lavage fluid and phospholipids

Pulmonary delivery appears to be the most promising non-parenteral route of insulin administration. In this work, we investigated the enhancement of insulin absorption in the presence of phospholipids and lung lavage fluid in vivo and in vitro. In-vitro experiments of insulin uptake by type II cells showed a significantly enhanced absorption in presence of lavage fluid, compared to various buffer preparations. The same trend was obtained with in-vivo studies of tracheal instillation of insulin. The incorporation of phospholipids as absorption enhancers in 1,2-dipalmitoyl phosphatidylcholine (DPPC) dispersion was compared to blank liposomes. A significantly higher blood glucose decrease was observed with a DPPC-insulin physical mixture compared to liposome, suggesting a possible effect of the phospholipid chain physical state on the insulin in-vivo absorption.     

The temporal profile of cytokines in the bronchoalveolar lavage fluid in mice exposed to the industrial gas phosgene

Diagnosis of an exposure to airborne toxicants can be problematic. Phosgene is used widely in industry for the production of many synthetic products, such as polyfoam rubber, plastics, and dyes. Although nearly 100% of the gas is consumed during processing, there is the potential problem of accidental or even intentional exposure to this irritant/choking agent. Exposure to phosgene has been known to cause latent life-threatening pulmonary edema. A major problem is that there is a clinical latency phase from 3 to 24 h in people before irreversible acute lung injury occurs. Assessment of markers of acute lung injury after a suspected exposure would be useful in developing rational treatment strategies. These experiments were designed to assess bronchoalveolar lavage fluid (BALF) for the presence of the early markers of exposure to phosgene in mice from 1 to 72 h after exposure. Separate groups of 40 CD-1 male mice (Crl:CD-1(ICR)BR) weighing 29 +/- 1 g were exposed whole-body to either air or a concentration x time (c x t) amount of 32 mg/m(3) (8 ppm) phosgene for 20 min (640 mg x min/m(3)). BALF from air- or phosgene-exposed mice was taken at 1, 4, 8, 12, 24, 48, and 72 h postexposure. After euthanasia, the trachea was excised, and 800 micro l saline was instilled into the lungs and washed 5x. BALF was assessed for interleukin (IL)-4, IL-6, tumor necrosis factor (TNF) alpha, IL-1alpha, macrophage inflammatory protein (MIP)-2, and IL-10. At 4 h postexposure, IL-6 was 15-fold higher for phosgene-exposed mice than for the time-matched air-exposed control group. At 8 and 12 h, IL-6, IL-1beta, MIP-2, and IL-10 were significantly higher in phosgene-exposed mice than in time-matched air-exposed controls, p < or = 0.05 to p < or = 0.001, whereas TNF alpha reached peak significance from 24 to 72 h. IL-4 was significantly lower in the phosgene-exposed mice than in the air-exposed mice from 4 to 8 h after exposure. These data show that BALF is an important tool in assessing pro- and anti-inflammatory markers of phosgene-induced acute lung injury and that knowledge of these temporal changes may allow for timely treatment strategies to be applied.     

Amiodarone — induced changes in surfactant phospholipids of rat lung

Summary Amiodarone HCl (AD) is a very effective antiarrhythmic drug, but its use is often associated with serious pulmonary complications. It is shown to induce lung phospholipidosis. Nevertheless, the effects of this drug on pulmonary surfactant which is composed of about 75% phospholipids and which prevents alveolar collapse is not known. Therefore, we have examined the effect of AD on the intra- and extracellular surfactant pools and on the levels of phosphatidylcholine (PC), the primary constituent of pulmonary surfactant.Male Wistar rats were fed AD (175 mg/kg) by oral gavage for three weeks. At the end of the experimental period, the rats were killed, the lungs removed and perfused, and surfactant isolated. Some lungs were prepared for ultrastructural examination. Phospholipid was assayed in the intra- and extracellular surfactant.Amiodarone produced a significant increase in both the intra- and extracellular sufactant phospholipid along with an appreciable change in the phospholipid profile. Also, the drug seemed to increase the number of lamellar inclusions in the surfactant producing type II alveolar cells. These data suggest that administration of AD leads to an increase in the lung surfactant phospholipid levels and lamellar bodies in alveolar type II cells.Correspondence to N. Devaraj at the above address     

Retention of inhaled perfluoroisobutene in the rat.

Perfluoroisobutene (PFIB) is produced by the pyrolysis, and as a by-product during the manufacture, of polytetrafluoroethylene. When inhaled it produces a fulminating and sometimes fatal pulmonary oedema similar to that of phosgene after a latent period of 6-8 h. As part of a study to determine the retained dose and the factors that control the amount retained, this study has investigated the retention in rats of inhaled PFIB at concentrations of 10, 50 and 250 micrograms l-1 in a flow-through system combining head-only exposure and plethysmography. Uptake of PFIB was measured by gas chromatography during elevated and reduced inspired volume and respiratory rate induced by exposure to increased CO2 and injection of pentobarbitone, respectively. The percentage of PFIB retained in the upper airways and lungs was found to be 27.5, 28.1 and 23.7% of the amount inspired at the three concentrations tested. The rate of uptake (nmol min-1 kg-1) of PFIB was a power law of the amount inhaled, an n-fold increase in minute volume producing an nb-fold increase in uptake, where b varied between 0.4 and 0.85. Thus, doubling the inhaled dose produces a 1.3-1.8-fold increase in uptake with a corresponding decrease in percentage retained. The relative contribution of respiratory rate and tidal volume upon PFIB retention could not be defined.     

The effect of lead exposure on some inflammatory biomarkers of lung lavage fluid in rats

This study was designed to examine the association between lead exposure and inflammatory responses in the lung lavage of rats. Thirty rats were randomly allocated into control (group C), sensitized (group S), and three exposed to lead (Pb) (groups 0.1M Pb and 0.4M Pb). Animal were sensitized with i.p. injected and aerosolized ovalbumin (OA). Pb-exposed groups inhaled 0.1M and 0.4M lead acetate for 1?h, thrice a week for two weeks. The levels of total protein, IgE and histamine were measured in the lung lavage of rats. The results of the present study showed significant increase in the levels of total protein, IgE and histamine in the lung lavage of rats exposed to high lead concentration and that of total protein and histamine in animal exposed to low lead concentration compared to control animals. The similar findings were also observed in sensitized animals. In addition, the levels of IgE and histamine in lung lavage of group exposed to high lead concentration significantly increased compared to group exposed to low lead concentration. The findings of the present study showed that exposure to inhaled lead acetate may lead to asthma like disease via inducing inflammatory responses in the lung lavage of rats.     

Influence of potential inhalation carriers on stability of thymopentin in rat bronchoalveolar lavage fluid

Abstract

In the present study, the stability of thymopentin (TP5) in bronchoalveolar lavage fluid (BALF) in presence of potential excipients in inhalation formulation was investigated. The content of TP5 was determined using HPLC method. Commonly used bulking agent, dispersibility enhancers and absorption enhancers in inhalation were investigated with respect to the stability of TP5 in BALF. Finally, the stability of TP5 in two inhalation formulations based on the screening experiments was tested in BALF. The results showed that TP5 alone degraded very rapidly in BALF and zero-order enzymatic reaction with a half-life of t_0.5?=?49.20?min was observed using 10 times diluted BALF. Among the amino acids examined, leucine and phenylalanine effectively inhibited the enzymolysis of TP5 with prolonged half-life of 112.7?min and 136.2?min, respectively. Nevertheless, slight but insignificant inhibition effect was witnessed for tyrosine, aspartic acid, and lysine; and negligible prevention on the degradation process of TP5 were found for lactose and mannitol. Regarding chitosan, irrespective with molecular weight, the formation of chitosan-TP5 complex improved the stability of TP5 with prolonged t_0.5 by 1.8 times. However, along with the improved stability of TP5 in spray-dried chitosan microspheres, the content of TP5 in formulations was reduced to about 75% during preparation process. Thus, leucine was proved to be a prior candidate for inhalation formulation of TP5. Consequently, the results indicate the potential of leucine as carrier for pulmonary delivery of TP5 serving as both stabilizer and dispersibility enhancer.     

The effect of phospholipids on anaphylactic histamine release

1. Histamine release by antigen from three sensitized rat tissues is potentiated by phosphatidyl serine (PS). The effect is greatest with isolated peritoneal cells. Phosphatidyl inositol, ethanolamine, choline and phosphatidic acid are inactive.

2. PS greatly increases the rate of antigen-induced histamine release and only slightly prolongs the duration of the release process.

3. PS shows a concentration-dependent effect over the range 1-10 μg/ml. At 10 μg/ml it is active over a wide range of antigen concentrations.

4. Calcium ions are necessary for the potentiation by PS of antigen-induced histamine release from rat peritoneal cells.

     

Reduction of hexavalent chromium by ascorbic acid in rat lung lavage fluid

The reduction of hexavalent chromium [chromium(VI)] in lung lavage fluids, microsomal (S–9) fractions of lung and liver tissues, erythrocyte lysates and plasma prepared from adult rats was examined at pH 7.4 (37° C). Specific reducing capacity, which was defined as the amount of chromium(VI) reduced per mg of protein in the test sample, was highest in the lavage fluids. The concomitant trivalent species [chromium(III)] was detected as complexes with some of the lavage components and probably as colloidal hydroxides. By gel filtration analysis and UV spectrometry, ascorbic acid (AsA) was identified as an important reducing factor in the lavage fluids. AsA levels in the lavage fluids were about 38 g/g tissue, corresponding to 12% of total AsA in the intact lungs. The molar ratios of oxidized AsA and reduced chromium(VI) in the lavage samples were about 32.3 on an average. On the basis of this molar ratio, the AsA levels in the lavage fluids are equivalent to a reducing capacity of 8.4 g chromium(VI)/g tissue. These results suggest that the lining layers (surfactant layers) of rat lungs provide an AsA-related capacity for protection of the cells against the toxic effects of chromates and probably other oxidants.     

The effect of phenobarbital and carbon tetrachloride on fatty acid content and composition of phospholipids from rat liver

The administration of phenobarbital or carbon tetrachloride to rats caused various changes in hepatic fatty acid content and composition. Phenobarbital elicited no effect on the total amount of fatty acids but significantly decreased myristic, pentadecanoic, and arachidonic acids and increased eicosatrienoic, eicosapentenoic, lignoceric, and docosatrienoic acid. In contrast, carbon tetrachloride enhanced significantly the total content and several components such as pentadecanoic, palmitic, palmitoleic, oleic, linoleic, arachidic, eicosenoic, eicosadienoic, eicosatrienoic, docosapentenoic, lignoceric and docosahexenoic acids. It elicited no effect on arachidonic acid. Unsaturated fatty acid moieties participating in the structure of these phosphatides were increased by phenobarbital and diminished by carbon tetrachloride. Phenobarbital caused a reduction in the ratio of saturated/unsaturated fatty acids mainly because of the decreased palmitic and increased oleic, linoleic, eicosatrienoic, arachidonic, docosapentenoic, and docosahexenoic acids. The significant variation brought about by phenobarbital and carbon tetrachloride on tissue fatty acids and in particular on fatty acid composition of phosphatidylcholine and phosphatidylethanolamine fractions reflects the opposing effects of these compounds on the liver cell. The major action of phenobarbital and carbon tetrachloride is associated with changes of the endoplasmic reticulum. Thus, their contrasting effect on fatty acid composition and metabolism may suggest that the disposition of lipid constituents plays a determinant role in the hepatic action of foreign compounds.     

盐酸氨溴索支气管肺泡灌洗联合肺表面活性物质对新生儿胎粪吸入综合征的治疗效果

目的探讨盐酸氨溴索支气管肺泡灌洗联合肺表面活性物质(PS)治疗新生儿胎粪吸入综合征(MAS)的疗效及安全性。方法 65例MAS患儿随机分为治疗组(A组,32例)和对照组(B组,33例),分别在治疗0、24、72h检测两组患儿支气管肺泡灌洗液(BALF)中肺表面活性蛋白(SP)A、SP-D及血管内皮生长因子(VEGF)的含量,并同时记录呼吸机参数、血气分析结果和呼吸机使用时间等临床指标。结果治疗24、48h后,A组患儿BALF中SP-A、SP-D含量逐渐升高,高于B组(P<0.05),而VEFG含量低于B组(P<0.05)。与B组相比,A组气胸发生例数、病死率、机械通气时间、氧暴露时间、住院天数均明显减少(P<0.05)。结论盐酸氨溴索支气管肺泡灌洗联合PS治疗MAS能通过促进内源性PS生成,使SP-A、SP-D含量增加,抑制VEGF的表达,从而减轻肺损伤,改善了氧合,并缩短了机械通气和氧暴露的时间,降低了病死率。     

Effects of paraquat on canine bronchoalveolar lavage fluid

Bronchoalveolar lavage (BAL) recovers the epithelial lung fluid of the lower respiratory tract. In this study, we have used BAL to detect early pulmonary injury in beagle dogs following an intravenous infusion of 10 mg paraquat dichloride/kg bodyweight. Bronchoalveolar lavage was performed twice in 11 dogs, 60 hr before and 34 hr after an intravenous infusion of paraquat dichloride (n = 8) or saline (n = 3). The dogs were studied in three groups: (1) paraquat only (n = 4); (2) paraquat plus hemoperfusion (n = 4); and (3) hemoperfusion only (n = 3). Because hemoperfusion, a treatment used for paraquat poisoning, could have effects on BAL independent of paraquat, we evaluated the effects on BAL fluid of this procedure performed separately from and together with administration of paraquat. We examined cytology, proteins, enzymes, and glutathione in the BAL fluid and expressed all results per milliliter of aspirated lavage fluid. Hemoperfusion did not alter the BAL fluid. In contrast, in dogs studied 34 hr after administration of paraquat, total cell counts, alveolar macrophage and neutrophil counts, and concentrations of total protein, albumin, ACE, LDH, and ALP were increased. Bronchoalveolar lavage in the dog provides an excellent tool with which to detect early paraquat-induced pulmonary injury. The same technique could be useful for sequential monitoring of other types of pulmonary disease and injury.     

The effect of loperamide oxide on prostaglandin-stimulated fluid transport in rat small intestine

The effects of loperamide and loperamide oxide on basal and prostaglandin E2-stimulated fluid transport by rat small intestine have been investigated. In contrast to loperamide, loperamide oxide, when applied intraperitoneally, failed to inhibit either basal or prostaglandin E2-stimulated fluid transport. However, intraperitoneal administration of loperamide oxide following its incubation with the contents of the intestinal lumen under aerobic conditions resulted in an effective inhibition of fluid secretion. The activating material was present in the essentially non-particulate 3000 g supernatant fraction of the luminal contents and was heat-stable.     

肺泡表面活性物质灌洗的浓度和时间对急性呼吸窘迫综合征治疗效果的影响

目的 观察肺泡表面活性物质(SF)灌洗的浓度和时间对急性呼吸窘迫综合征(ARDS)治疗效果的影响.方法 用0.0225N盐酸12 ml/kg注入新西兰白兔气管内,建立ARDS模型后随机分成七组,每组5只.在盐酸注入后1h用浓度为1、3、6和12g/L的SF肺灌洗,2和3h用浓度为12 g/L的SF肺灌洗;对照组用生理盐水灌洗.观察治疗前后二氧化碳分压(PaCO2)和气道吸气峰压(PIP)变化;并对肺标本行病理检查.结果 造模1h后,3、6和12 g/L的SF灌洗均明显降低PaCO2,作用持续1.5h;造模2h后用浓度为12 g/L灌洗组PaCO2降低作用可持续2h.但3h组PaCO2未见降低.各灌洗组均不能降低PIP.结论 盐酸造模后2h内进行SF 3～12 g/L肺灌洗可改善肺换气功能.     

Characterisation of the rat lung lavage model as biological assay for testing the activity of surfactant preparations

The present report describes how pharmacological assays may be validated and sets a basis for a discussion on the validation of biological test systems. The note for guidance on the validation of analytical procedures published by the European agency for the evaluation of medicinal products was adapted to the validation of a pharmacological test system. The presently described rat lung lavage test (RLL-test) is an animal model that has great similarities to the pathophysiology of the acute respiratory distress syndrome of humans. In this RLL-test, the activity of surfactants can be tested in a standardised fashion. The usefulness of the point estimator and the corresponding confidence intervals (CI) as a statistical test procedure for equivalence was demonstrated. A validation can be based on the above mentioned guidance but should be adjusted to pharmacological needs. Based on the presented experiences, it can be concluded that a specific guideline for validation of pharmacological or biological tests is desirable.     

The effect of auranofin on the colonic transport of Na+ and fluid in the rat

Auranofin in the mucosal fluid caused a dose-dependent inhibition of fluid and Na+ absorption by everted sacs of rat colon. Serosal auranofin was without effect. (Na+ + K+)ATPase activity of homogenates of mucosal scrapes of rat colon was inhibited by auranofin in a dose-related manner, while Mg2+-ATPase activity was little affected. These actions of the drug on colonic transport mechanisms could contribute to the diarrhoea associated with auranofin therapy.     

Contribution of C-fibers to leucocyte recruitment in bronchoalveolar lavage fluid and pleural cavity in the rat

The effect of neonatal capsaicin (8 methyl-N-vanillyl-6-nonenamide) treatment on the leucocyte infiltration into the airways and pleural cavity was investigated in rats actively sensitized with ovalbumin. The animals were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or vehicle (10% ethanol and 10% Tween 80). At adult ages, the animals were actively sensitized with ovalbumin (200 microg, s.c.) and 14 days later they were intratracheally (or intrapleurally) challenged with ovalbumin. The substance P level in bronchoalveolar lavage fluid of the capsaicin group was reduced by >90% compared to control group (vehicle), confirming the efficacy of capsaicin treatment. In the capsaicin group, the number of neutrophils (but not of eosinophils and mononuclear cells) in bronchoalveolar lavage fluid of sensitized animals was significantly higher than the control group. Intrapleural injection of ovalbumin in sensitized rats caused a significant neutrophil influx at 6 h that was markedly increased in the capsaicin-pretreated animals compared to control group. The counts of eosinophils and mononuclear cells in the pleural exudates did not differ significantly between capsaicin and control groups. The increased levels of immunoglobulin (Ig)E, IgG1 and IgG2a anti-ovalbumin antibodies in serum of sensitized rats did not differ between capsaicin and control groups. In conclusion, the exacerbated pulmonary neutrophil recruitment caused by the capsaicin neonatal treatment is unrelated to increase in serum immunoglobulin antibodies, and suggests a protective role for C-fibers in attenuating the allergic neutrophil infiltration.     

The kallikrein kinin system in pulmonary lavage fluid of reserpinized rats

Kallikrein content in lavage of the respiratory tract was determined by a specific RIA. The results show an immunological identity of the lung kallikrein with the standard rat urinary kallikrein. The levels of immunoreactive lung fluid kallikrein were significantly lower in rats injected with reserpine.