Ventilated Rabbit Lungs Research Articles

Suppression of pulmonary group B streptococcal proliferation and translocation by surfactants in ventilated near-term newborn rabbits.

The pathogenesis of neonatal group B Streptococcus (GBS) lung infection may be associated with surfactant dysfunction or deficiency. This study aimed to investigate the efficacy of surfactants on early postnatal GBS infection in ventilated newborn rabbit lungs. A near-term newborn rabbit model was established by intratracheal GBS instillation immediately at birth, followed by mechanical ventilation. At postnatal 1 h, a porcine surfactant was given intratracheally at 100 or 200 mg/kg. After 6 h, animals were euthanized, and lung and blood samples were collected for bacterial counting. Lung histopathology and messenger RNA (mRNA) expression of inflammatory mediators, surfactant proteins, and growth factors in lung tissue were assessed. The surfactants significantly suppressed (by >50%) pulmonary bacterial proliferation and systemic translocation, alleviated lung inflammatory injury, and improved alveolar expansion by morphometry, in favor of high-dose surfactants. Though the survival rate and lung mechanics were not improved, the surfactants significantly suppressed mRNA expression of proinflammatory mediators, while that for surfactant proteins and growth factors was differentially expressed, compared to the control and GBS infection groups. Exogenous surfactants may provide a therapeutic alternative for neonatal lung infection by suppressing pulmonary GBS proliferation and translocation into systemic circulation, alleviating inflammatory injury and regulating growth factor expression.

Acute histopathological effects of cryotherapy on lung tissue

Background: Cryotherapy is an actual treatment and diagnostic method in many fields of medicine. We aimed to evaluate the acute histopathological effects of cryotherapy application on lung tissue. Materials and Methods: In this study 10 male rabbits were used. After providing single lung ventilation of the rabbits, clamshell incision was performed for exploration. Four different cryotherapy applications were performed to determine the different histopathologic changes on atelectatic and ventilated lung, also the difference between one versus two-cycle applications. After all applications, firstly, air leak was checked, then specimens were taken to analyze the acute histopathologic effects. Results: After cryotherapy application, no air leak was detected on ventilated rabbit lung with a pressure of 30 cmH2O. There was no bleeding from lung tissue, but macroscopic parenchymal hematoma developed. The depth of necrosis in lung tissue was evaluated with histopathological analysis. In all samples, alveolar edema and congestion were observed but there was no statistical difference between the depth of necrosis regarding with different cryotherapy applications. Conclusions: This study indicated that different applications of cryotherapy on lung parenchyma is a safe method and do not affect the depth of necrosis in a statistically significant degree.

Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

BackgroundAcute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined.MethodWe employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability.ResultsIn hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W.ConclusionHypercapnia with and without acidosis increased HPV during conditions of sustained hypoxia. The increase of sustained HPV and endothelial permeability in hypoxic hypercapnia without acidosis was iNOS dependent.

Positive effects of oxygenation on simulated post-transplantation lung edema and pulmonal arterial resistance

Objective: Ischemia-reperfusion injury still causes severe problems after lung transplantation. It is supposed that systemic oxygen supply to the transplanted lungs could improve transplantation outcome. By using an ex vivo model of isolated perfused and ventilated rabbit lungs we investigated the influence of systemic oxygenation on pulmonary function during simulated transplantation. Methods: The heart-lung blocks of 12 New Zealand White rabbits (2.22±0.4kg) were excised and transferred to a 37°C water-heated artificial thorax chamber. Negative pleural pressure ventilation was initiated and perfusion with recirculating, albumin-containing Tyrode-Solution was slowly increased to a constant end flow of 100ml/min. The lungs were arranged in a stable perfusion and ventilation mode for 30min. Afterwards lungs underwent a flush-perfusion with Perfadex® Solution, followed by an ischemic storage for 4h on ice, thereafter ventilation and reperfusion for 2h were continued to simulate a transplantation situation (oxygenated group, pulmonary artery pO2=120mmHg). In another series the perfusate inflow was additionally gassed with nitrogen (connecting a fiber oxygenator) to simulate deoxygenation (deoxygenated group, pulmonary artery pO2=50mmHg). Hemodynamic and ventilatory parameters (pulmonary artery pressure (PAP), pulmonary compliance, pulmonary resistance, lung weight) were continuously detected. Results: After 2h reperfusion time in the oxygenated group we found a significant lower PAP and lung weight compared to the deoxygenated group (p<0.05). PAP and lung weight steadily increased after reestablishment of lung perfusion (PAP: initial 8.78±0.89 cmH2O, 30min reperfusion 7.2±1.48 cmH2O, 2h reperfusion 11.5±1.06 cmH2O; lung weight: initial 22.1±1.32g, 30min reperfusion 26.1±1.87g, 2h reperfusion 35.4±4.23g). This development was significantly influenced by the intravascular pO2 (PAP: initial 9.65±0.43 cmH2O, 30min reperfusion 7.92±0.52 cmH2O, 2h reperfusion 8.02±0.63 cmH2O; lung weight: initial 17.9±1.54g, 30min reperfusion 19.0±1.68g, 2h reperfusion 21.5±2.29g, p<0.05). Conclusions: Oxygenation of the lung perfusate during simulated transplantation attenuates post transplant edema formation and decreases pulmonal arterial hypertension. Transfering this to the surgical situation, revascularisation of bronchial arteries after lung transplantation might initiate positive effects in the early phase after lung transplantation.

Pretreatment with perfluorohexane vapor attenuates fMLP-induced lung injury in isolated perfused rabbit lungs

ABSTRACTThe authors investigated the protective effects and dose dependency of perfluorohexane (PFH) vapor on leukocyte-mediated lung injury in isolated, perfused, and ventilated rabbit lungs. Lungs received either 18 vol.% (n = 7), 9 vol.% (n = 7), or 4.5 vol.% (n = 7) PFH. Fifteen minutes after beginning of PFH application, lung injury was induced with formyl-Met-Leu-Phe (fMLP). Control lungs (n = 7) received fMLP only. In addition 5 lungs (PFH-sham) remained uninjured receiving 18 vol.% PFH only. Pulmonary artery pressure (mPAP), peak inspiratory pressure (Pmax), and lung weight were monitored for 90 minutes. Perfusate samples were taken at regular intervals for analysis and representative lungs were fixed for histological analysis. In the control, fMLP application led to a significant increase of mPAP, Pmax, lung weight, and lipid mediators. Pretreatment with PFH attenuated the rise in these parameters. This was accompanied by preservation of the structural integrity of the alveolar architecture and air-blood barrier. In uninjured lungs, mPAP, Pmax, lung weight, and lipid mediator formation remained uneffected in the presence of PFH. The authors concluded that pretreatment with PFH vapor leads to an attenuation of leukocyte-mediated lung injury. Vaporization of perfluorocarbons (PFCs) offers new therapeutic options, making use of their protective and anti-inflammatory properties in prophylaxis or in early treatment of acute lung injury.

Effects of perfluorohexane vapor in the treatment of experimental lung injury

Rationale We investigated the effects of vaporized perfluorohexane (PFH) on pulmonary vascular tone, pulmonary vascular resistance and peak inspiratory pressure as well as lipid mediator formation in the treatment of calcium ionophore induced lung injury in a model of the isolated perfused and ventilated rabbit lungs. Methods Lung injury was induced in isolated perfused and ventilated rabbit lungs by calcium ionophore A23187. Lungs were treated with either 4.5 vol.% (4.5 vol.% PFH; n = 6) or 18 vol.% (18 vol.% PFH; n = 6) PFH. Six lungs remained untreated (Control). In addition 5 lungs (PFH-sham) remained uninjured receiving 18 vol.% PFH only. Mean pulmonary artery pressure (mPAP), peak inspiratory pressure ( P max), and lung weight (weight) were monitored for 120 min. Experiments were terminated before when the increase in lung weight exceeded 40 g. Perfusate samples were taken at regular intervals for analysis of TXB 2, 6-keto-PGF 1 and LTB 4. Results Controls reached the study end point significantly earlier than both PFH groups. Significant differences were found for a weight gain of 10 g and 20 g between the control and the 4.5 vol.% PFH and the 18 vol.% PFH. Differences in mPAP were more pronounced in the 4.5 vol.% PFH. However increases in P max were more marked in 4.5 vol.% PFH. TXA 2-, PGI 2-, and LTB 4-levels were significantly lower in PFH groups. Uninjured lungs remained unaffected by the presence of 18 vol.% PFH. Conclusion Inflammatory lung injury was attenuated by the treatment with 4.5 vol.% PFH and 18 vol.% PFH vapor in the isolated perfused rabbit lung. Therapeutic effects were more pronounced with a concentration of 4.5 vol.% PFH.

Inducible nitric oxide synthase inhibition attenuates lung injury and decreases NADPH oxidase expression and activation during ischemia‐reperfusion in the ventilated rabbit lung

Lung ischemia‐reperfusion (IR) causes arteriolar constriction and platelet adhesion in correlation with inducible nitric oxide synthase (iNOS) expression/activity and reactive nitrogen species (RNS). While iNOS was elevated during reperfusion, RNS were only elevated in lungs that underwent ischemia. To determine oxidative stress, and if attenuation of vascular dysfunction by iNOS inhibition could be due to decreased NADPH oxidase stimulation, we assessed p47 phox translocation and vascular leakage. In anesthetized rabbits with open chest, we ventilated the lungs with an oxygen mixture and examined subpleural microvessels and alveoli in the right lung during brief periods when the lungs were statically inflated (10 cm H2O), IR was induced by occluding the right pulmonary artery for 2 h. Fluorescently labeled albumin was injected via the right atrium to assess vascular leakage. Oxidative stress was assessed by translocation of the cytosolic NADPH oxidase subunit, p47 phox, to the membrane fraction by immunoblotting. Vascular leakage and translocation of p47 phox were greater in reperfused ventilated lungs than in contralateral or control lungs, and were attenuated by pretreatment with an iNOS activity inhibitor (1400W). Our data suggest that stimulation of NADPH oxidase is an important component in acute lung injury in conjunction with iNOS overexpression. Supported by ALA of KY and NIH grant # HL080394.

Direct eicosanoid profiling of the hypoxic lung by comprehensive analysis via capillary liquid chromatography with dual online photodiode-array and tandem mass-spectrometric detection

Eicosanoids are arachidonic acid-derived mediators, with partly contradictory, incompletely elucidated actions. Thus, epoxyeicosatrienoic acids (EETs) are controversially discussed as putative vasodilatative endothelium-derived hyperpolarizing factors in the cardiovascular compartment but reported as vasoconstrictors in the lung. Inconsistent findings concerning eicosanoid physiology may be because previous methods were lacking sensitivity, identification reliability, and/or have focused on special eicosanoid groups only, ignoring the overall mediator context, and thus limiting the correlation accuracy between autacoid formation and bioactivity profile. Therefore, we developed an approach which enables the simultaneous assessment of 44 eicosanoids, including all representatives of the arachidonic acid cascade, i.e., cytochrome P450, lipoxygenase, cyclooxygenase products, and free isoprostanes as in vivo markers of oxidative stress, in one 50-minute chromatographic run. The approach combines (i) source-specific sample extraction, (ii) rugged isocratic and high-sensitivity capillary liquid-chromatographic separation, and (iii) reliable dual online photodiode-array and electrospray ionization tandem mass-spectrometric identification and quantitation. High sensitivity with limits of quantification in the femtogram range was achieved by use of capillary columns with typical high peak efficiency, due to small inner diameters, and virtually complete substance transfer to the mass spectrometer, due to flow rates in the low microliter range, instead of large inner diameter columns with low chromatographic signal and only partial analyte transfer employed by previous methods. This expeditious, global and sensitive technique provides the prerequisite for new, accurate insights regarding the physiology of specific mediators, for example EETs, in the context of all relevant vasoactive autacoids under varying conditions of oxidative stress by direct comparison of all eicosanoid generation profiles. Indeed, application of comprehensive "eicoprofiling" to hypoxically ventilated rabbit lungs revealed at a glance the enhanced biosynthesis of free EETs in the overall mediator generation context, thus suggesting their hypothetical contribution to hypoxic pulmonary vasoconstriction.

Pulmonary absorption of aerosolized fluorescent markers in the isolated rabbit lung

For the development of aerosolized controlled release formulations such as liposomes or nanoparticles, the use of suitable model drugs is necessary. This study compared the pulmonary absorption of the three structurally diverse fluorescent markers 5(6)-carboxyfluorescein (CF), 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt (MPTS) and rhodamine 6G (R6G) after nebulization in an isolated, perfused and ventilated rabbit lung. Aerosol particle size and lung deposition as well as lipophilicity of the fluorescent markers were determined. Dye concentrations were measured in the recirculating buffer and in the bronchoalveolar lavage. The MMAD of the dye aerosols ranged between 4.70 and 4.88 μm, total lung deposition was 0.40 ± 0.05 ml. The 1-octanol/water partition coefficient as measure for lipophilicity was −3.45 ± 0.16 for CF, −4.95 ± 0.21 for MPTS and 2.69 ± 0.18 for R6G. The perfusate concentration showed an increase to ∼400 ng/ml (53.4 ± 6.8% of the intrapulmonary deposited dye) for CF and ∼230 ng/ml (29.1 ± 2.0%) for MPTS, respectively; R6G concentration increased in the first 30 min to ∼38 ng/ml followed by a gradual decrease to ∼26 ng/ml (3.3 ± 0.7%). In conclusion, these data suggest that the hydrophilic dye CF is suitable to study drug transport from aerosolized controlled release formulations across the lung barrier. In contrast, the highly water-soluble fluorescent probe MPTS demonstrates insufficient recovery and the lipophilic R6G high accumulation in lung tissue.

Adenosine A1 and A2 receptor agonists reduce endotoxin-induced cellular energy depletion and oedema formation in the lung

Tissue depletion of adenosine during endotoxaemia has previously been described in the lung. Therapeutic approaches to prevent adenosine depletion and the role of A1 and A2 receptor agonists, however, have not been investigated until now. In isolated and ventilated rabbit lungs, it was tested whether pretreatment with adenosine A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 10(-7) mol, n = 6) or A2 receptor agonist 5'-(N-cyclopropyl)-carboxyamido adenosine (CPCA; 10(-7) mol, n = 6) prior to injection of lipopolysaccharide (LPS) (500 pg mL-1) influenced pulmonary artery pressure (PAP), pulmonary energy content and oedema formation as compared with controls, solely infused with LPS (n = 6). Release rates of adenosine and uric acid were determined by high-performance liquid chromatography. Pulmonary tissue concentrations of high-energy phosphates were measured and the adenine nucleotide pool, adenosine 5'-triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio and adenylate energy charge of the pulmonary tissue were calculated. Administration of LPS induced increases in PAP within 2 h up to 20.8 +/- 2.9 mmHg (P < 0.01). While pretreatment with the A1 agonist merely decelerated pressure increase (13.8 +/- 1.1 mmHg, P < 0.05), the A2 agonist completely suppressed the pulmonary pressure reaction (9.6 +/- 1.0 mmHg, P < 0.01). Emergence of lung oedema after exclusive injection of LPS up to 12.0 +/- 2.9 g was absent after A1 (0.6 +/- 0.5 g) and A2 (-0.3 +/- 0.2 g) agonists. These observations were paralleled by increased adenosine release rates compared with LPS controls (P < 0.05). Moreover, tissue concentrations of ADP, ATP, guanosine 5'-diphosphate, guanosine 5'-triphosphate, nicotinamide-adenine-dinucleotide and creatine phosphate were significantly reduced after LPS. Consequently, the calculated tissue adenine nucleotide pool and the adenylate energy charge increased after adenosine receptor stimulation (P = 0.001). Adenosine A1- and A2-receptor agonists reduced LPS-induced vasoconstriction and oedema formation by maintenance of tissue energy content. Thus, adenosine receptor stimulation, in particular of the A2 receptor, might be beneficial during acute lung injury.

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

BackgroundThe sources and measurement of reactive oxygen species (ROS) in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR) with spin trapping is a specific method for ROS detection, and may address some these technical problems.MethodsWe have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe l-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway.ResultsWhile perfusing lungs with CPH over a range of inspired oxygen concentrations (1–21 %), the rate of CP• formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD) to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA) into the pulmonary artery caused a rapid increase in CP• formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5 % O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases.ConclusionThe perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to demonstrate that 1) PMA-induced vasoconstriction is caused "directly" by superoxide generated from NADPH oxidases and 2) this pathway is pronounced in hypoxia. NADPH oxidases thus may contribute to the hypoxia-dependent regulation of pulmonary vascular tone.

Evaluation of particle translocation across the alveolo-capillary barrier in isolated perfused rabbit lung model

Particulate air pollution is associated with respiratory and cardiovascular morbidity and mortality. It has been suggested that ultrafine particles are able to translocate from the airways into the bloodstream in vivo. We have investigated this in an isolated perfused and ventilated rabbit lung preparation lacking pulmonary lymphatic flow. Fluorescent polystyrene particles of different diameters (24, 110 or 190 nm) and surface chemistry (carboxylate or amine modified) were injected either intratracheally (i.t.) or intravascularly (i.v.) and, after a period of 2 h, their presence in the perfusion liquid or in the bronchoalveolar lavage (BAL) fluid, was assessed by spectrofluorimetry. Vascular pressures and lung weights were monitored. Following the i.t. administration, no particle translocation was observed from the alveoli into the vascular compartment. Similarly, no particle translocation was found after i.v. administration of particles. However, when microvascular permeability was pharmacologically increased by administering histamine (10(-4) M) in the vascular compartment, inducing a positive driving force provided by fluid filtration, a fluorescent signal in BAL was recorded (2.5 +/- 1% of the dose of particles administered), suggesting a translocation of particles through the alveolo-capillary barrier. We conclude that ultrafine polystyrene particles cannot significantly diffuse from lung into the vascular compartment in our model, but they are able to translocate in the opposite direction when the microvascular permeability is increased by histamine. The relevance of these ex vivo findings for the in vivo translocation of inhaled ultrafine particles remains to be established.

Hyperinflation during lung preservation and increased reperfusion injury 1

Reperfusion injury after lung transplantation remains a perplexing and unpredictable problem. Most surgeons preserve the lung inflated, but the amount of inflation that should be used is not well documented. Therefore, we studied the effect of high inflation during organ preservation on lung function during reperfusion. Our hypothesis is that donor lung hyperinflation during storage contributes to early allograft dysfunction during reperfusion. To test our hypothesis we used an isolated, blood-perfused, ventilated rabbit lung model. Group I lungs (control) underwent immediate reperfusion after harvest. Group II lungs (low-inflation, maintained at 6 mmHg airway pressure) and group III lungs (high-inflation, maintained at 20 mmHg airway pressure) were stored for 4 h in 4 degrees C Euro-Collins solution after harvest. All lungs were then reperfused with whole blood for 1 h, and measurements of arterial oxygenation (PO2, mmHg), pulmonary artery pressure (PAP, mmHg), peak inspiratory pressure (PIP, cm H2O), and wet-to-dry weight ratio (WTD) were obtained. Throughout the 1 h reperfusion period group III lungs had significantly lower oxygenation compared to groups I and II. In addition, throughout reperfusion, group III lungs showed significantly higher PAP and PIP compared to group II. WTD did not differ significantly between groups, however, there was a trend toward increased edema in group III. These results indicate that high inflation during cold storage results in acute pulmonary dysfunction. Careful monitoring of airway inflation pressure during storage, especially to prevent hyperinflation, should be maintained in the current practice for lung transplantation.

Increase in alveolar antioxidant levels in hyperoxic and anoxic ventilated rabbit lungs during ischemia

Increases in free radicals are believed to play a central role in the development of pulmonary ischemia/reperfusion (I-R) injury, leading to microvascular leakage and deterioration of pulmonary surfactant. Continued ventilation during ischemia offers significant protection against I-R injury, but the impact of alveolar oxygen supply both on lung injury and on radical generation is still unclear. We investigated the influence of hyperoxic (95% O 2) and anoxic (0% O 2) ventilation during ischemia on alveolar antioxidant status and surfactant properties in isolated rabbit lungs. Normoxic and hyperoxic ventilated, buffer-perfused lungs ( n = 5 or 6) and native lungs ( n = 6) served as controls. As compared with controls, biophysical and biochemical surfactant properties were not altered in anoxic as well as hyperoxic ventilated ischemic (2, 3, and 4 h) lungs. Assessment of several antioxidants (reduced glutathione (GSH), α-tocopherol (vitamin E), retinol (vitamin A), ascorbic acid (vitamin C), uric acid, and plasmalogens (1- O-alkenyl-2-acyl-phospholipids)) in bronchoalveolar lavage fluid (BALF) revealed a significant increase in antioxidant compounds under anoxic and hyperoxic ventilation, with maximum levels occuring after 3 h of ischemia. For example, GSH increased to 5.1 ± 0.8 μM (mean ± SE, p < .001) after 3 h of anoxic ventilated ischemia and to 2.7 ± 0.2 μM ( p < .01) after hyperoxic ventilated ischemia compared with native controls (1.3 ± 0.2 μM), but did not significantly change under anoxic and hyperoxic ventilation alone. In parallel, under ischemic conditions, oxidized glutathione (GSSG) increased during hyperoxic (3 h: 0.81 ± 0.04 μM, p < .001), but remained unchanged during anoxic (3 h: 0.31 ± 0.04 μM) ventilation compared with native controls (0.22 ± 0.02 μM), whereas F 2-isoprostanes were elevated under both hyperoxic (3 h: 63 ± 15 pM, p < .01) and anoxic (3 h: 50 ± 9 pM, p < .01) ventilation compared with native controls (16 ± 4 pM). We conclude that oxidative stress is increased in the lung alveolar lining layer during ischemia, during both anoxic and hyperoxic ventilation. This is paralleled by an increase rather than a decrease in alveolar antioxidant levels, suggested to reflect an adaptive response to oxidative stress during ischemia.

Clusterin protects the lung from leukocyte-induced injury.

Clusterin (CLU) is a multifunctional 75- to 80-kDa glycoprotein that is upregulated during cellular stress and might represent a defense mechanism during local cellular damage. Mechanisms discussed are antiapoptotic, antioxidative, and anticomplement properties as well as chaperone-like features protecting stressed proteins. The aim of this study was to investigate potential protective effects of CLU on pulmonary vasculature after in situ PMN activation in isolated rabbit lungs. The experiments were performed on 24 isolated and ventilated rabbit lungs that were perfused with 200 mL of Krebs-Henseleit-10% blood buffer with a constant flow of 150 mL/min in a recirculating system. It was tested whether pretreatment with CLU (2.5 microg/ml; n = 8) or catalase (CAT, 5000 U/ml; n = 8) before N-formyl-Met-Leu-Phe (fMLP; 10(-8) M) injection influenced pulmonary artery pressure (PAP) peak airway pressures (PAW) and edema formation as compared with controls (n = 8). Baseline values of PAP were 9-11 mmHg and PAW 11-13 cm H2O. Application of fMLP resulted in an acute significant (P < 0.01) increase of PAP (48 +/- 29 mmHg) within 2 min in the control group and PAW increased to 35 +/- 7 cm H2O within 30 min. Pretreatment with CLU completely suppressed the PAP and PAW response as a result of the fMLP challenge (P < 0.001), whereas a transient PAW increase up to 27 +/- 15 mmHg was observed after CAT. Complement factor C3a release was suppressed by CAT, whereas CLU blocked the complement cascade at the level of C5b-9 formation. Moreover, generation of thromboxane A(2) was reduced after CLU and CAT. Lung edema occurred in the fMLP group but was absent (P < 0.001) after CLU and CAT treatment. Both CLU and CAT prevented fMLP-induced lung injury. Stabilizing effects of CLU, point towards complement regulating features at the level of the terminal complement sequence. Elevated levels of CLU during inflammation could reflect a compensatory organ protective mechanism. Further studies are required to elucidate the clinical impact of the observed organ-protective properties of CLU.

Warm ischemia lung protection with pinacidil: an ATP regulated potassium channel opener

Background Ischemia/reperfusion injury remains a limiting factor in lung transplantation. Traditional hyperkalemic preservation solutions are associated with a host of metabolic derangements. ATP-regulated potassium channel openers (PCOs) may provide an attractive alternative to traditional solutions by utilizing inherent mechanisms of ischemic preconditioning. The purpose of this study was to assess warm ischemia graft protection with pinacidil, a nonspecific PCO. Methods An isolated recirculating blood perfused ventilated rabbit lung model was used (n = 15). No ischemia control lungs underwent immediate reperfusion (n = 5). Warm ischemia control lungs were flushed with lactated Ringers (LR), stored at 37°C for 2.5 hours and then reperfused for 2 hours (n = 5). PCO protected lungs were flushed with LR + 100 μmol/L pinacidil, stored, and then reperfused (n = 5). Intermittent blood gases were taken from the pulmonary artery and left atria. Every 30 minutes, graft function was assessed with a 10-minute 100% fractional inspired oxygen concentration challenge to measure maximal gas exchange. Lung samples were graded for histologic injury and assayed for myeloperoxidase activity. Results A mixed-models repeated measures ANOVA demonstrated a significant difference between groups. Tukey’s honestly significant difference multiple comparison test demonstrated significantly improved graft function and reduced histologic injury with pinacidil protection compared with the warm ischemia controls. There was no significant difference in graft function or pathology grade between the pinacidil protected lungs and the no ischemia controls. A similar trend, although not significant, was seen in myeloperoxdiase activity. Conclusions Potassium channel openers with pinacidil can provide pulmonary protection against warm ischemia reperfusion injury.

Novel protection strategy for pulmonary transplantation

Background. Ischemia-reperfusion injury continues to represent a significant challenge to successful lung transplantation. Traditional pulmonary ischemic protection is performed using hypothermic hyperkalemic depolarizing solutions to reduce the metabolic demands of the ischemic organ. Measures to further reduce the effects of ischemic injury have focused on the reperfusion period. We tested the hypothesis that novel physiologic hyperpolarizing solutions—using ATP-dependent potassium channel (K ATP) openers—given at the induction of ischemia, will reduce cellular injury and provide superior graft function even after prolonged periods of ischemia. Methods. An isolated blood-perfused ventilated rabbit lung model was used to study lung injury. Airway, left atrial, and pulmonary artery pressures were measured continuously during the 2-h reperfusion period. Oxygenation, as a surrogate of graft function, was measured using intermittent blood gas analysis of paired left atrial and pulmonary artery blood samples. Graft function was measured by oxygen challenge technique (F iO 2 = 1.0). Wet-to-dry ratio was measured at the conclusion of the 2-h reperfusion period. Control (Group I) lungs were perfused with modified Euro-Collins solution (depolarizing) and reperfused immediately (no ischemia). Traditional protection lungs were perfused with modified Euro-Collins flush solution and stored for 4 h (Group II) or 18 h (Group III) at 4°C before reperfusion. Novel protection (Group IV) lungs were protected with a hyperpolarizing solution containing 100 nM Aprikalim, a specific K ATP channel opener, added to the modified Euro-Collins flush solution and underwent 18 h of ischemic storage at 4°C before reperfusion. Results. Profound graft failure was measured after 18 h of ischemic storage with traditional protection strategies (Group III). Graft function was preserved by protection with hyperpolarizing solutions even for prolonged ischemic periods (Group IV). Wet-to-dry weight ratio, airway, left atrial, and pulmonary artery pressures were not significantly different between the groups. Conclusions. We have created a model of predictable lung injury. Membrane hyperpolarization with a K ATP channel opener (PCO) provides superior prolonged protection from ischemia-reperfusion injury in an in vitro model of pulmonary transplantation.

Interaction of acetylcholine and endothelin-1 in the modulation of pulmonary arterial pressure.

The study was designed to investigate the effects of acetylcholine (ACh) on pulmonary circulation with special regard to mediators that could be involved in the mediation of ACh-induced effects. ACh has been reported to induce either vasodilation or vasoconstriction in the pulmonary circulation of different species. Prospective experimental study in rabbits. Experimental laboratory in a university teaching hospital. Sixty-six adult rabbits of either sex. The experiments were performed on 66 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. ACh was injected in various concentrations after pulmonary artery preconstriction and in untreated lungs. Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. Perfusate samples were taken intermittently to determine endothelin-1 (ET-1), thromboxane A2 (TXA2), and prostacyclin (PGI2) concentrations. ACh in final dosages from 10(-5) to 10(-2) M (n = 6 each) was injected into the pulmonary artery of lungs treated with U46619 to induce pulmonary arterial hypertension or was injected into untreated lungs. To analyze the potential mechanisms of action, ACh (10(-5) M) was administered in additional experiments after pretreatment with either ETA receptor antagonist BQ123 (10(-6) M; n = 6) or the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6). In preconstricted pulmonary vessels, ACh (10(-3) and 10(-2) M) initially induced a PAP rise for 10 mins followed by a sustained decrease. In untreated lungs, ACh induced an immediate dose-dependent increase in PAP, requiring as long as 30 mins to return to predrug levels. Simultaneously, significantly elevated TXA2 and PGI2 levels were observed. Furthermore, ET-1 was detected in the perfusate, which was free from ET-1 before ACh administration. Pretreatment with BQ123 reduced substantially the ACh (10(-5) M)-induced PAP increase and the release of TXA2 and PGI2. At 5 mins, the PAP maximum was reduced from 18.5 +/- 3.2 mm Hg to 9.9 +/- 0.65 mm Hg by BQ123 pretreatment (p < .01). An inhibition of PAP increase was also observed after diclofenac pretreatment (11.6 +/- 0.4 mm Hg at 5 mins; p < .05). Inhibitory effects at 5 mins were significantly more pronounced in the BQ123 group compared with the diclofenac group. The effects of ACh on the pulmonary circulation of isolated rabbit lungs depend on ACh concentration and the basal tone of the arterial vasculature. In lungs with a normal pulmonary vascular resistance, ACh administration causes vasoconstriction via the release of ET-1 and TXA2, whereas vasodilation is induced in preconstricted pulmonary vessels.

Aprotinin improves pulmonary function during reperfusion in an isolated lung model

Background. We hypothesized that the use of aprotinin would ameliorate the reperfusion injury observed after lung transplantation because of a reduction in the inflammatory response. Methods. We used an isolated, whole blood-perfused, ventilated rabbit lung model to study the effects of aprotinin during reperfusion. The control animals (group A, n = 8) underwent lung harvest after pulmonary arterial prostaglandin E 1 injection and Euro-Collins preservation flush before saline storage for 18 hours at 4°C. The experimental groups received either a low dose (3,000 KIU/mL; group B, n = 8) or a high dose (10,000 KIU/mL; group C, n = 8) of aprotinin added to the pulmonary flush before storage. Each lung was reperfused at 37°C at a rate of 60 mL/min. Results. The arterial partial pressure of oxygen values of group B (low-dose aprotinin) were significantly higher than those of group A (control) after 10 minutes of reperfusion (69.19 ± 5.69 mm Hg versus 264.30 ± 48.59 mm Hg, respectively, p = 0.001). Similar results were recorded at 20 and at 30 minutes of reperfusion. Similarly, after 10 minutes of reperfusion, the differences between groups A and C were 69.19 ± 5.69 mm Hg versus 235.91 ± 28.63 mm Hg, respectively ( p = 0.001). Conclusions. The addition of aprotinin to the Euro-Collins pulmonary flush significantly improves arterial oxygenation in the early reperfusion period. The enhanced oxygenation suggests that aprotinin may offer protection against early reperfusion injury.

Impact of endothelin-1 in endotoxin-induced pulmonary vascular reactions.

Elevated endothelin-1 (ET-1) levels have been detected during sepsis. The aim of the study was to examine the role of thromboxane A2 (TXA2) and ET-1 in pulmonary vascular reactions after endotoxin (LPS) challenge. Prospective experimental study in rabbits. Experimental laboratory in a university teaching hospital. Twenty-four adult rabbits of either sex. Experiments were performed on 30 isolated and ventilated rabbit lungs, which were perfused with a saline solution containing 10% autologous blood. Pulmonary arterial pressure and lung weight gain were continuously registered. Perfusate samples were drawn intermittently to determine ET-1, TXA2, and prostacyclin (PGI2) concentrations. LPS isolated from Escherichia coli (0.5 mg/mL; n = 6) was added to the perfusate. A marked pulmonary arterial pressure increase followed by massive edema formation after 60 mins was observed after LPS injection. At the same time, elevated TXA2 and PGI2 levels in the perfusate were measured. ET-1 was detected 30 mins after LPS infusion (13.4+/-2.6 fmol/L). Pretreatment with the ET(A) receptor antagonist LU135252 (10(-6) M; n = 6) almost completely suppressed the pressure reaction after endotoxin injection (p < .01 at 50 and 60 mins) and reduced edema formation (p < .05). The cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) was as effective as LU135252 in preventing vascular reactions after LPS injection. Pretreatment with the ET(A) receptor antagonist LU135252 and the cyclooxygenase inhibitor diclofenac reduced pulmonary vascular reactions after LPS challenge. Based on the current data, we conclude that the pulmonary arterial pressure increase and edema formation after LPS injection are related to an ET-1- and TXA2-dependent mechanism.