H. Neuhof

Increased pulmonary vascular resistance and permebility due to arachidonate metabolism in isolated rabbit lungs

Liberation and metabolism of arachidonic acid may be the common final pathway of different stimuli on the pulmonary vascular bed. In a model of isolated, ventilated rabbit lungs, perfused with Krebs Henseleit albumin buffer in a recirculating system, changes of pulmonary vascular resistance and of vascular permeability are monitored continuously. The addition of free arachidonic acid or of the Ca-ionophore A 23187 to the perfusion fluid consistently evokes a biphasic increase in vascular resistance as well as an initially reversible increase in vascular permeability, followed by pulmonary edema. Both phases of increased vascular resistance are completely suppressed by inhibition of the cyclooxygenase, decreased to a large degree by inhibitors of thromboxane synthetase, and markedly augmented by short preincubation of arachidonic acid with ram seminal vesicular microsomes and by sulfhydryl reagents. The increased pulmonary vascular permeability is augmented by inhibition of cyclooxygenase and reduced by simultaneous lipoxygenase inhibition. Antagonists of histamine, serotonin and sympathic or parasympathic activity do not have any influence. PG F2alpha., TxB2, PG E2 and PG I2 alter the pulmonary vascular resistance, but do not increase vascular permeability. In conclusion, increased availability of free arachidonic acid evokes a rise in pulmonary vascular resistance, which can be ascribed to cyclooxygenase products, especially to thromboxane, and causes a rise in vascular permeability which can be ascribed to lipoxygenase products. The findings may be related to acute pulmonary lesions with increase in vascular resistance and with vascular leakage.

Alteration of alveolar surfactant function after exposure to oxidative stress and to oxygenated and native arachidonic acid in vitro

UNLABELLED Alveolar surfactant is known to be impaired after inhalation of various oxidizing agents (NO2, ozone) as well as in inflammatory lung processes, in which leucocyte-derived active oxygen species or arachidonic acid oxygenation products may be involved. The effect of lipid peroxidation, oxygen-free radicals and oxygenated versus native arachidonic acid on the surface tension behaviour of natural surfactant was tested in vitro. The studies were performed on pooled surfactant material, obtained from bronchoalveolar lavage of rabbit lungs, in a Langmuir trough/Wilhelmy balance system. Initiation of lipid peroxidation with FeCl3/ascorbate or UV radiation and the generation of OH.(FeCl2/EDTA/H2O2), O2-. (xanthine/xanthine oxidase) and 1O2 (NaOCl/H2O2) provoked a common profile of changes: delayed reduction of surface tension during compression with an increase in minimal compressibility accelerated decrease of film pressure during expansion, reduction of hysteresis area and markedly augmented monolayer collapse rate. Addition of arachidonic acid resulted in decreased minimal compressibility, stability index and hysteresis area. Incubation with the arachidonic acid cyclooxygenase products, prostaglandin E2, I2, F2 alpha or thromboxane B2, with soybean lipoxygenase or with H2O2 and O2-exposure caused only moderate or no alteration of surfactant behaviour in vitro. CONCLUSION oxidative stress, but not arachidonic acid oxygenation products, provoked altered surface tension behaviour of natural surfactant in vitro.

Actions and interactions of mediator systems and mediators in the pathogenesis of ARDS and multiorgan failure.

A great variety of mediators and mediator systems are involved in the disturbance of the microcirculation and vascular permeability following polytrauma and sepsis. The locally accentuated, organ related activation and the cooperation of several of these mediators and mediator systems over a longer period of time seem to be responsible for the development of an acute organ failure in terms of ARDS and MOF. Cytokines from macrophages seem to be the determining factors converting a primarily functional and reversible systemic vascular reaction into organ related morphological lesions. This pathogenetic complexity has to be considered in future concepts for therapy and prophylaxis with regard to the hierarchical rank of the mediators involved.

Glutathione redox cycle is an important defense system of endothelial cells against chronic hyperoxia

Exposure of cultured pulmonary artery endothelial cells to 95% O2 resulted in the following sequence of events: decrease in [3H]thymidine incorporation after 24 h; increase of intracellular glutathione (GSH) and loss of cellular protein after 48 h; increase of spontaneous and decrease of provoked prostacyclin formation as well as increased release of cellular LDH after 72 h. This oxygen toxicity model was used to study the following 2 questions. (1) What is the relative importance of the GSH redox cycle compared to catalase as antioxidative defense against hyperoxia? Endothelial cells were grown in selenium-depleted medium to inhibit glutathione peroxidase activity. Endothelial GSH biosynthesis was inhibited by buthionine sulfoximine. Catalase activity was reduced by aminotriazole. Endothelial cells with an impaired GSH redox cycle were easily killed by hyperoxia within 24 h, while inhibition of catalase did not enhance the susceptibility of endothelial cells to hyperoxia. (2) Can endothelial GSH content be increased by exogenous sulfhydryl reagents and does this results in an increase of endothelial cells’ resistance to hyperoxia? Exogenous GSH, N-acetylcysteine, cysteine, and L-2-oxothiazolidine-4-carboxylate (L-2-oxo) increased intracellular GSH. All sulfhydryl reagents (with the exception of L-2-oxo) protected endothelial cells from hyperoxia. Concentrations of exogenous GSH and N-acetylcysteine that did not increase intracellular GSH reduced hyperoxia-induced endothelial cell injury. Thus the capacity of the GSH redox cycle rather than intracellular GSH levels or catalase determines endothelial cells’ resistance to hyperoxia.

Alterations of bacterial clearance induced by propofol

Background: The purpose of the study was to investigate the potential influence of the anaesthetic agent propofol on immune function in terms of systemic clearance and organ distribution of injected Escherichia coli in a rabbit model.

Effects of N-acetylcysteine on bacterial clearance

Abstract. The aim of this study was to investigate whether the oxygen radical scavenger N‐acetylcysteine (N‐AC) impairs bacterial clearance, thus predisposing the host to increased risk of disease. Blood clearance of Escherichia coli and organ colonization were investigated in anaesthetized rabbits after pretreatment with N‐AC (250 mg kg‐1 body weight, n= 16) and in sham‐operated animals (n= 12). To enable quantification of the clearance process, defined numbers of exogenous E. coli [1.3 times 108 colony‐forming units (CFUs)] were injected intravenously. Parameters monitored were kinetics of bacterial elimination from the blood, and polymorphonuclear leucocyte (PMN) oxidative burst activity. Samples of liver, kidney, spleen and lung were collected for bacterial counts. Compared with controls, pretreatment with N‐AC resulted in delayed bacterial elimination from blood and higher organ colonization with increased numbers of E. coli in liver, lung and kidney (P < 0.05). N‐AC treatment was associated with a suppressed PMN oxidative burst activity. Impaired bacterial clearance and enhanced organ colonization in N‐AC‐treated animals correlated with reduced oxidative burst activity, suggesting impaired granulocyte‐dependent bacterial killing due to N‐AC application.

Calpain system and its involvement in myocardial ischemia and reperfusion injury.

Calpains are ubiquitous non-lysosomal Ca(2+)-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca(2+)-content and Ca(2+)-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.

Endotoxemia and cytokine generation in cardiac surgery in relation to flow mode and duration of cardiopulmonary bypass.

ABSTRACT We investigated whether pulsatile flow in cardiopulmonary bypass (CPB), which has been shown to improve intestinal perfusion, reduces endotoxin translocation from the gut and, in consequence, decreases cytokine generation. The study population consisted of 48 adult patients who underwent elective CPB surgery. Pulsatile flow was used during aortic cross‐clamping in 24 patients and nonpulsatile flow in 24 patients. Plasma endotoxin concentration increased in all patients during CPB. Significantly (P < 0.05) lower peak levels of 8.25 ± 1.17 (SEM) pg/mL were reached 30 min after CPB in patients with pulsatile flow in contrast to 11.26 ± 1.42 pg/mL in patients with nonpulsatile flow. The extent of endotox‐ emia was not related to the duration of CPB. Following the increase of plasma endotoxin, the concentrations of IL‐6 and IL‐8 increased with delay of approximately 1 h. The peak levels of these cytokines corresponded significantly (P < 0.005 and P < 0.01, respectively) with duration of CPB, but not with flow mode. Thus, in patients with CPB of more than 97 min (median), IL‐6 reached a peak of 335.5 ± 48.87 pg/mL and IL‐8 of 64.86 ± 24.79 pg/mL in contrast to 210.9 ± 18.45 pg/mL and 21.2 ± 10.19 pg/mL, respectively, with bypass times of less than 97 min. The degree of endotoxemia in CPB mainly depends on the quality of tissue perfusion. Cytokine generation, however, is not triggered exclusively by endotoxin, but rather by the trauma of CPB and surgery.

Endothelin-1 and thromboxane A2 increase pulmonary vascular resistance in granulocyte-mediated lung injury.

OBJECTIVE To examine the pathophysiologic role of vasoactive eicosanoids and endothelin-1 in granulocyte-mediated effects in the pulmonary vasculature. DESIGN Prospective experimental study in rabbits. SETTING Experimental laboratory in a university teaching hospital. SUBJECTS Thirty adult rabbits. INTERVENTIONS The experiments were performed on 30 isolated and ventilated rabbit lungs that were perfused with a cell- and plasma-free buffer solution. MEASUREMENTS AND MAIN RESULTS The pulmonary arterial pressure and the lung weight gain were continuously registered. Intermittently perfused samples were taken to determine endothelin-1 and thromboxane A2 concentrations. Six experiments without intervention served as the sham group. The granulocytes in the pulmonary circulation were stimulated with N-formyl-L-leucin-methionyl-L-phenylalanine (FMLP; 10(-6) M; control, n = 6). To investigate whether activated granulocytes influence the pulmonary vasculature via endothelin-1, the endothelin-A receptor antagonist LU135252 (10(-6) M) was added to the perfusate before FMLP injection (n = 6). The potential involvement of thromboxane A2 in granulocyte-endothelial interaction was investigated by pretreatment with the cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6). Activation of granulocytes resulted in an acute increase in pulmonary arterial pressure (>9 mm Hg), which was followed by a second delayed pressure increase after 60 mins (>14 mm Hg) and was paralleled by a massive generation of thromboxane A2 (>250 pg/ mL). Fifteen minutes after FMLP-injection, endothelin-1 was detectable in the perfusate. Pretreatment with the selective endothelin-A antagonist LU135252 significantly (p< .01) reduced the initial pressure response after FMLP stimulation, while diclofenac significantly reduced (p < .05) the delayed pressure increase. Using diclofenac (10 microg/mL) in conjunction with LU135252 (10(-6) M; n = 6) before FMLP injection significantly reduced the early and the delayed pressure increase. CONCLUSIONS Activated granulocytes seem to enhance pulmonary vascular resistance via endothelin-1 and thromboxane A2. The endothelin-1 effects are probably mediated via endothelin-A receptors since the endothelin-A receptor antagonist LU135252 was able to suppress the early pressure reaction after FMLP injection, whereas the cyclooxygenase inhibitor diclofenac was able to reduce the second pressure increase.

Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts

Abstract Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9±2.3% of the area at risk in untreated controls (n=12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10-8 M A-705253 (n=8), which reduced (p<0.001) the infarcted area to 49.3±3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.