Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C

INTRODUCTION: Studies in vitro reveal that endothelin-1 (ET-1) activates the α isoform of protein kinase C (PKC-α) in cultures of endothelial cells, thereby deranging cellular integrity. Sepsis and endotoxemia are associated with increased plasma concentrations of ET-1 that induce acute lung injury (ALI). We recently reported that non-selective ET-1 receptor blockade attenuates ALI in sheep by reducing the endotoxin-induced increase in extravascular lung water index (EVLWI). The aim of this study was to find out whether this attenuation is associated with reduced translocation of PKC-α from the cytosolic to the membrane fraction of lung tissue homogenate. METHODS: Seventeen awake, instrumented sheep were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide (LPS) group (n = 7) receiving an intravenous infusion of Escherichia coli 15 ng/kg per min for 24 hours, and a tezosentan group (n = 7) subjected to LPS and, from 4 hours, an intravenous injection of tezosentan 3 mg/kg followed by infusion at 1 mg/kg per hour for the reminder of the experiment. Pulmonary micro-occlusion pressure (Pmo), EVLWI, plasma concentrations of ET-1, tumor necrosis factor-a (TNF-a), and interleukin-8 (IL-8) were determined every 4 hours. Western blotting was used to assess PKC-α. RESULTS: In non-treated sheep a positive correlation was found between the plasma concentration of ET-1 and Pmo in the late phase of endotoxemia (12 to 24 hours). A positive correlation was also noticed between Pmo and EVLWI in the LPS and the LPS plus tezosentan groups, although the latter was significantly reduced in comparison with LPS alone. In both endotoxemic groups, plasma concentrations of ET-1, TNF-α, and IL-8 increased. In the LPS group, the cytosolic fraction of PKC-α decreased by 75% whereas the membrane fraction increased by 40% in comparison with the sham-operated animals. Tezosentan completely prevented the changes in PKC-α in both the cytosolic and the membrane fractions, concomitantly causing a further increase in the plasma concentrations of ET-1, TNF-α, and IL-8. CONCLUSION: In endotoxemic sheep, ET-1 receptor blockade alleviates lung injury as assessed by a decrease in EVLWI paralleled by a reduction in Pmo and the prevention of activation of PKC-α

An easy "SteamDrop" method for high quality plant chromosome preparation

BACKGROUND: The chromosome preparation is a crucial step for obtaining satisfactory results in molecular cytogenetic researches. The preparation of plant chromosomes for molecular cytogenetic purposes remains a challenge for some species. In contrast to human chromosome preparation, the processes occurring during plant chromosome preparation and causing chromosome spreading are still poorly understood. RESULTS: We studied the dynamics of plant chromosome spreading after dropping cell suspension on slides. We showed that steam stimulates cytoplasm hydrolysis and rapid chromosome spreading and that chromosomes stretch during this chromosome spreading. Based on these observations, we developed a novel method, named “SteamDrop”, for the preparation of well-spread mitotic and pachytene chromosomes and successfully used it for 28 plant species with large and small chromosomes. We applied cell suspensions in ethanol instead of the commonly used ethanol/acetic acid fixative. Mitotic and meiotic chromosomes prepared via “SteamDrop” were used in fluorescent in situ hybridization (FISH) experiments with repetitive and unique DNA probes. Long storage of cell suspensions in ethanol did not impair the quality of chromosome preparations. CONCLUSION: The SteamDrop procedure provides a robust and routine method for high quality plant chromosome preparations. The method can be applied for metaphase as well as pachytene chromosome preparation in wide range of species. The chromosomes prepared by SteamDrop are well suitable for repetitive and unique DNA visualization

Measuring extravascular lung water: animals and humans are not the same

The evolution of extravascular lung water (EVLW) monitoring is an important step forward in the hemodynamic assessment of critically ill patients

Recombinant human activated protein C ameliorates oleic acid-induced lung injury in awake sheep

Introduction: Acute lung injury (ALI) may arise both after sepsis and non-septic inflammatory conditions and is often associated with the release of fatty acids, including oleic acid (OA). Infusion of OA has been used extensively to mimic ALI. Recent research has revealed that intravenously administered recombinant human activated protein C (rhAPC) is able to counteract ALI. Our aim was to find out whether rhAPC dampens OA-induced ALI in sheep. Methods: Twenty-two yearling sheep underwent instrumentation. After 2 days of recovery, animals were randomly assigned to one of three groups: (a) an OA+rhAPC group (n = 8) receiving OA 0.06 mL/kg infused over the course of 30 minutes in parallel with an intravenous infusion of rhAPC 24 mg/kg per hour over the course of 2 hours, (b) an OA group (n = 8) receiving OA as above, or (c) a sham-operated group (n = 6). After 2 hours, sheep were sacrificed. Hemodynamics was assessed by catheters in the pulmonary artery and the aorta, and extravascular lung water index (EVLWI) was determined with the single transpulmonary thermodilution technique. Gas exchange was evaluated at baseline and at cessation of the experiment. Data were analyzed by analysis of variance; a P value of less than 0.05 was regarded as statistically significant. Results: OA induced profound hypoxemia, increased right atrial and pulmonary artery pressures and EVLWI markedly, and decreased cardiac index. rhAPC counteracted the OA-induced changes in EVLWI and arterial oxygenation and reduced the OA-induced increments in right atrial and pulmonary artery pressures. Conclusions: In ovine OA-induced lung injury, rhAPC dampens the increase in pulmonary artery pressure and counteracts the development of lung edema and the derangement of arterial oxygenation

Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep

INTRODUCTION: Acute lung injury is associated with accumulation of extravascular lung water (EVLW). The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLW(ST)) and postmortem gravimetric (EVLW(G)). METHODS: Eighteen instrumented and awake sheep were randomly assigned to one of three groups. All groups received Ringer's lactate (5 ml/kg per hour intravenously). To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours (n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min (n = 7). A third group (n = 4) was subjected to sham operation. Haemodynamic variables, including EVLW(ST), were measured using a PiCCOplus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLW(ST )was compared with EVLW(G). RESULTS: At the end of experiment, values for EVLW(ST )(mean ± standard error) were 8.9 ± 0.6, 11.8 ± 1.0 and 18.2 ± 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively (P < 0.05). The corresponding values for EVLWI(G )were 6.2 ± 0.3, 7.1 ± 0.6 and 11.8 ± 0.7 ml/kg (P < 0.05). Ranges of EVLWI(ST )and EVLWI(G )values were 7.5–21.0 and 4.9–14.5 ml/kg. Regression analysis between in vivo EVLW(ST )and postmortem EVLW(G )yielded the following relation: EVLW(ST )= 1.30 × EVLW(G )+ 2.32 (n = 18, r = 0.85, P < 0.0001). The mean bias ± 2 standard deviations between EVLW(ST )and EVLW(G )was 4.9 ± 5.1 ml/kg (P < 0.001). CONCLUSION: In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry

Recombinant human activated protein C attenuates endotoxin-induced lung injury in awake sheep

Introduction: Acute lung injury often complicates severe sepsis. In Gram-negative sepsis, bacterial endotoxin activates both coagulation and inflammation. Enhanced lung vascular pressures and permeability, increased extravascular lung water content and deteriorated gas exchange characterize ovine endotoxin-induced lung injury, a frequently used model of acute lung injury. Recombinant human activated protein C (rhAPC), with its anticoagulant, anti-inflammatory, fibrinolytic and antiapoptotic effects, reportedly reduces the respiratordependent days and the mortality of patients with severe sepsis. We speculate whether rhAPC antagonizes endotoxin-induced lung injury in sheep. Methods: Two groups of sheep were exposed to Escherichia coli endotoxin (lipopolysaccharide) 15 ng/kg/minute intravenously from 0 to 24 hours; one group received only lipopolysaccharide throughout (n = 8), and the other group received lipopolysaccharide in combination with rhAPC 24 μg/ kg/hour from 4 to 24 hours (n = 9). In addition, one group received rhAPC as above as the only intervention (n = 4), and four sham-operated sheep were used for determination of the α and ε isoforms of protein kinase C in pulmonary tissue. Data were assessed by one-way analysis of variance for repeated measurements. Biochemical data were analyzed using Student's t test, or using the Mann–Whitney U test when appropriate. Results: Infusion of endotoxin caused lung injury, manifested by increments in pulmonary artery pressure, in pulmonary microocclusion pressure, in pulmonary vascular downstream resistance, in pulmonary vascular permeability index, in extravascular lung water index and in deterioration of oxygenation that were all attenuated by infusion of rhAPC. Endotoxemia led to changes in inflammation and coagulation, including pulmonary neutrophil accumulation paralleled by increased TNFα and decreased protein C and fibrinogen in animal plasma, which all improved following infusion of rhAPC. Moreover, rhAPC prevented the translocation of protein kinase C α and ε isoforms from the cytosolic fraction of lung tissue extracts. Conclusion: In awake sheep, rhAPC alleviates endotoxininduced lung injury – as characterized by improvements of oxygenation, coagulation and inflammation, as well as by reversal of pulmonary hemodynamic and volumetric changes

Increased Extravascular Lung Water Reduces the Efficacy of Alveolar Recruitment Maneuver in Acute Respiratory Distress Syndrome

Introduction. In acute respiratory distress syndrome (ARDS) the recruitment maneuver (RM) is used to reexpand atelectatic areas of the lungs aiming to improve arterial oxygenation. The goal of our paper was to evaluate the response to RM, as assessed by measurements of extravascular lung water index (EVLWI) in ARDS patients. Materials and Methods. Seventeen adult ARDS patients were enrolled into a prospective study. Patients received protective ventilation. The RM was performed by applying a continuous positive airway pressure of 40 cm H2O for 40 sec. The efficacy of the RM was assessed 5 min later. Patients were identified as responders if PaO2/FiO2 increased by >20% above the baseline. EVLWI was assessed by transpulmonary thermodilution before the RM, and patients were divided into groups of low EVLWI (<10 mL/kg) and high EVLWI (≥10 mL/kg). Results. EVLWI was increased in 12 patients. Following RM, PaO2/FiO2 increased by 33 (4–65) % in the patients with low EVLWI, whereas those in the high EVLWI group experienced a change by only −1((−13)–(+5)) % (P = 0.035). Conclusion. In ARDS, the response to a recruitment maneuver might be related to the severity of pulmonary edema. In patients with incresed EVLWI, the recruitment maneuver is less effective

Inhaled aerosolised recombinant human activated protein C ameliorates endotoxin-induced lung injury in anaesthetised sheep

Introduction We recently demonstrated that intravenously infused recombinant human activated protein C (APC) attenuates ovine lipopolysaccharide (LPS)-induced lung injury. In this study, our aim was to find out whether treatment with inhaled aerosolised APC (inhAPC) prevents formation of increased lung densities and oedema and derangement of oxygenation during exposure to LPS. Methods: Sheep were anaesthetised during placement of intravascular introducers. After one to four days of recovery from instrumentation, the animals were re-anaesthetised, endotracheally intubated and mechanically ventilated throughout a six-hour experiment where the sheep underwent quantitative lung computed tomography. Sheep were randomly assigned to one of three groups: a sham-operated group (n = 8) receiving inhaled aerosolised saline from two hours after the start of the experiment; a LPS group (n = 8) receiving an intravenous infusion of LPS 20 ng/kg per hour and, after two hours, inhaled aerosolised saline over the next four hours; a LPS+inhAPC group (n = 8) receiving an intravenous infusion of LPS 20 ng/kg per hour and, after two hours, aerosolised APC 48 µg/kg per hour inhaled throughout the experiment. Data were analysed with analysis of variance; P less than 0.05 was regarded as significant. Results: An infusion of LPS was associated with a reduction of well-aerated lung volume and a rapid fall in arterial oxygenation that were both significantly antagonised by inhaled APC. Pulmonary vascular pressures and extravascular lung water index increased significantly during exposure to LPS, but inhaled APC had no effect on these changes. Conclusions: Inhalation of aerosolised APC attenuates LPSinduced lung injury in sheep by preventing a decline in the volume of aerated lung tissue and improving oxygenation

Features of Anesthesiologists-Reanimatologists’ Emotional States in Different COVID-19 Pandemic Periods in Russia

Introduction: The COVID-19 pandemic is one of the most stressful events in recent times. Medical professionals, including anesthesiologists-reanimatologists, suffered the main blow in this difficult and stressful environment. Aims: This study aimed at identifying the features of anesthesiologists-reanimatologists’ emotional states in different COVID-19 pandemic periods. Methods: The study was conducted through an anonymous questionnaire among anesthesiologists-reanimatologists in two periods. In the First stage – which was carried out in May 2020 (during the first COVID-19 pan¬demic wave) – 58 anesthesiologists-reanimatologists in the Arkhangelsk region took part. During the Second segment – which took place in October 2020 (in the second COVID-19 pandemic wave) – 43 anesthesiologists-reanimatologists were examined. Repeated questioning was carried out among the same participants. Results: In October 2020, compared to May, the number of doctors who noted a high intensity of professional activity increased. Regardless of the study period, one-third of the subjects experienced constant pronounced anxiety. Anesthesiologists-reanimatologists, whose professional activity was directly related to the patients in COVID-19 care, noted a poorer emo¬tional state more frequently in October, accompanied by anxiety, depressed mood, irritability and a high burnout level, which may indicate a depletion of internal resources in this group. Conclusions: The study results showed that for anesthesiologists-reanimatologists, a further depletion of emotional resources accompanied the second pandemic wave. The anesthesiologists-reanimatologists’ emotional state was mediated by a number of social and gender factors, as well as specific labor organization features

Comparison of Goal-Directed Hemodynamic Optimization Using Pulmonary Artery Catheter and Transpulmonary Thermodilution in Combined Valve Repair: A Randomized Clinical Trial

Our aim was to compare the effects of goal-directed therapy guided either by pulmonary artery catheter (PAC) or by transpulmonary thermodilution (TTD) combined with monitoring of oxygen transport on perioperative hemodynamics and outcome after complex elective valve surgery. Measurements and Main Results. Forty patients were randomized into two equal groups: a PAC group and a TTD group. In the PAC group, therapy was guided by mean arterial pressure (MAP), cardiac index (CI) and pulmonary artery occlusion pressure (PAOP), whereas in the TTD group we additionally used global end-diastolic volume index (GEDVI), extravascular lung water index (EVLWI), and oxygen delivery index (DO2I). We observed a gradual increase in GEDVI, whereas EVLWI and PAOP decreased by 20–30% postoperatively (P < 0.05). The TTD group received 20% more fluid accompanied by increased stroke volume index and DO2I by 15–20% compared to the PAC group (P < 0.05). Duration of mechanical ventilation was increased by 5.2 hrs in the PAC group (P = 0.04). Conclusions. As compared to the PAC-guided algorithm, goal-directed therapy based on transpulmonary thermodilution and oxygen transport increases the volume of fluid therapy, improves hemodynamics and DO2I, and reduces the duration of respiratory support after complex valve surgery