Massive and long-lasting decrease in vitamin C plasma levels as a consequence of extracorporeal circulation.

OBJECTIVE: The use of cardiopulmonary bypass (CPB) is suggested to induce oxidative stress, reflected by an imbalance between prooxidant and antioxidant substances. The majority of studies published have either focused on only one aspect (prooxidant or antioxidant side) or covered only a short observation period. Therefore, the aim of this study was to investigate the long-term effects of CPB on the balance of prooxidative markers and antioxidant substances in one single group of patients, being able to estimate the degree of oxidative stress. METHODS: Blood samples were taken from 29 patients undergoing cardiovascular surgery beginning the day before surgery through postoperative day 6 (discharge). Plasma concentrations of vitamins C (total ascorbic acid) and E and malondialdehyde were measured by high-performance liquid chromatography. Plasma levels of ascorbyl free radical were determined using electron paramagnetic resonance spectroscopy. RESULTS: The study showed a significant decrease in vitamin C plasma levels during CPB without any recovery of vitamin C up to the time of discharge. Furthermore, CPB induced a significant increase in malondialdehyde plasma concentrations immediately after unclamping, accompanied by a significant increase in the ascorbyl free radical to total ascorbic acid ratio. The latter stayed elevated until the end of observation. CONCLUSIONS: Our findings indicate that the oxidative stress event after CPB can be divided into two phases: Immediately after reperfusion, a massive oxidative stress occurs, reflected by the increase in malondialdehyde. During convalescence, there must be an ongoing situation of oxidative stress, especially in the water-soluble compartment, leading to the consumption of vitamin C. Because the main antioxidant substance, vitamin C, did not increase again over the entire observation period, supplementation should be given consideration.

Neurosurgical procedures in the semisitting position: evaluation of the risk of paradoxical venous air embolism in patients with a patent foramen ovale.

OBJECTIVE: To analyze the actual risk for patients with a patent foramen ovale (PFO) to experience a clinically relevant venous air embolism (VAE) during surgery performed in the semisitting position. METHODS: All procedures were performed between January 2008 and December 2009, under general anesthesia and in the semisitting position. Transesophageal echocardiography (TEE) and capnometry were used intraoperatively to monitor for air bubbles in the venous system. RESULTS: Of 200 consecutive patients who all were operated on in the semisitting position, 52 patients (26%) had a diagnosis of PFO. Rates of VAE in patients were graded as follows: grade 0 (no air bubbles visible, no air embolism), 23 patients (44.2%); grade I (air bubbles on TEE), 22 patients (42.3%); grade II (air bubbles on TEE with decrease of end-tidal carbon dioxide [ETCO2] ≤ 3 mm Hg), 2 patients (3.8%); grade III, air bubbles on TEE with decrease of ETCO2 >3 mm Hg, 4 patients (7.7%); grade IV, air bubbles on TEE with decrease of ETCO2 >3 mm Hg and decrease of mean arterial pressure ≥ 20% or increase of heart rate ≥ 40% (or both), 1 patient (1.9%); and grade V, VAE causing arrhythmia with hemodynamic instability requiring cardiopulmonary resuscitation, 0 patients (0%). There were no deaths in this series, and no new or unexplained, mild or severe neurologic deficits were caused by a VAE. CONCLUSIONS: Under standardized anesthesia and neurosurgical protocols, patients with a PFO can be operated on safely in the semisitting position.

Activated protein C ameliorates impaired renal microvascular oxygenation and sodium reabsorption in endotoxemic rats.

INTRODUCTION: We aimed to test whether continuous recombinant human activated protein C (APC) administration would be able to protect renal oxygenation and function during endotoxemia in order to provide more insight into the role of coagulation and inflammation in the development of septic acute kidney injury. METHODS: In anesthetized, mechanically ventilated Wistar rats, endotoxemia was induced by lipopolysaccharide administration (10 mg/kg i.v. over 30 min). One hour later, the rats received fluid resuscitation with 0 (LPS + FR group; n = 8), 10 (APC10 group; n = 8), or 100 (APC100 group; n = 8) µg/kg/h APC for 2 h. Renal microvascular oxygenation in the cortex and medulla were measured using phosphorimetry, and renal creatinine clearance rate and sodium reabsorption were measured as indicators of renal function. Statistical significance of differences between groups was tested using two-way ANOVA with Bonferroni post hoc tests. RESULTS: APC did not have notable effects on systemic and renal hemodynamic and oxygenation variables or creatinine clearance. The changes in renal microvascular oxygenation in both the cortex (r = 0.66; p < 0.001) and medulla (r = 0.80; p < 0.001) were correlated to renal sodium reabsorption. CONCLUSION: Renal sodium reabsorption is closely correlated to renal microvascular oxygenation during endotoxemia. In this study, fluid resuscitation and APC supplementation were not significantly effective in protecting renal microvascular oxygenation and renal function. The specific mechanisms responsible for these effects of APC warrant further study.

The volatile anesthetic sevoflurane inhibits activation of neutrophil granulocytes during simulated extracorporeal circulation.

Extracorporeal circulation (ECC) is an essential tool for the execution of cardiac operations. However, ECC is also associated with undesirable side effects. These include the induction of a systemic inflammatory response associated with leukocyte activation and cytokine release as well as potentially life-threatening complications. The volatile anesthetic sevoflurane has been reported to exert anti-ischemic and anti-inflammatory effects. We therefore investigated whether sevoflurane modulates the ECC-triggered inflammatory response. Heparinized human blood was circulated for 90 min in a normothermic (37°C) ex vivo ECC circuit. An air-oxygen mixture was administered via an oxygenator in controls (n=5). Sevoflurane (2 vol.%) was added to the gas mixture in a second group (n=5). At baseline and after 30, 60 and 90 min of ECC, blood samples were taken. In each sample whole blood counts were determined. Expression of the activation-indicating Mac-1 receptor on granulocytes and monocytes as well as leukocyte-platelet aggregate formation was measured in flow cytometry. Levels of the granulocyte activation marker PMN-elastase and of the cytokines IL-1ß, IL-8 and TNF-α were analyzed using ELISA. ECC induced significant increases in Mac-1 expression on granulocytes (p<0.001) and PMN-elastase release (p<0.001). Sevoflurane decreased granulocyte Mac-1 expression during ECC (p<0.05) and inhibited the ECC-induced PMN-elastase release (p<0.05). Sevoflurane had no effect on whole blood cell counts, leukocyte-platelet aggregate formation and cytokine release during ECC. Sevoflurane inhibits granulocyte activation during ex vivo ECC and therefore has the potential to decrease the ECC-triggered inflammatory response. This promising finding warrants further investigation under clinical conditions.

[Resuscitation algorithms for cardiac arrest and arrhythmias]. / Reanimation: Was ist neu? Notfallbehandlung von Herzstillstand und Arrhythmien.

Recently, guidelines for cardiopulmonary resuscitation have been updated by the European Resuscitation Council. Here we focus on cardiac arrest and arrhythmia algorithms for primary healthcare providers incorporating new recommendations.

ImageStream cytometry extends the analysis of phagocytosis and oxidative burst.

AIM: Phagocytosis is often measured using conventional microscopy and flow cytometry. ImageStream cytometry is a new technology that combines the advantages of both methods, enabling statistically robust microscopic applications. We compared ImageStream cytometry to flow cytometry in a whole blood model of phagocytosis with viable, fluorescence-marked Staphylococcus aureus. We furthermore measured the co-localization of intracellular bacteria to sites of oxidative burst, as well as changes in cell size and actin levels as a result of phagocytosis. EXPERIMENTAL DESIGN: Fluorescence-labeled S. aureus in a ratio of 5:1 bacteria per leukocyte were added to whole blood. Phagocytosis was stopped at different time points. After staining of neutrophils and lysis of erythrocytes, samples were analysed by ImageStream cytometry and flow cytometry. RESULTS: Phagocytosis and oxidative burst determined by flow cytometry and ImageStream cytometry showed strong correlation. In contrast to flow cytometry, ImageStream cytometry easily detected and excluded extracellular adherent bacteria from the measurement of phagocytosis, and enumerated the bacteria within each neutrophil. Using the Bright Detail Similarity score, we identified a subset of neutrophils with intracellular bacteria co-localized to sites of oxidative burst activity. Phagocytosis resulted in an increase in cell size and actin polymerization as determined by an increase in phalloidin fluorescence intensity. CONCLUSIONS: We describe a simple whole blood image-based method for measuring bacterial phagocytosis and oxidative burst. ImageStream cytometry provides the spatial resolution to determine the number of bacteria ingested and the sub-cellular localization and trafficking patterns that enables a more complete evaluation of the phagocytic process.

Cytomegalovirus reactivation and associated outcome of critically ill patients with severe sepsis.

INTRODUCTION: Sepsis has been identified as a risk factor for human cytomegalovirus (CMV) reactivation in critically ill patients. However, the contribution of CMV reactivation on morbidity and mortality is still controversial. Therefore, we analyzed the incidence and impact of CMV reactivation on outcome in patients with severe sepsis. METHODS: In a prospective longitudinal double-blinded observational study, 97 adult nonimmunosuppressed CMV-seropositive patients with new onset of severe sepsis were included. Leukocytes, plasma and tracheal secretions were examined weekly for CMV-DNA by PCR. Tracheal secretions were additionally tested for HSV (Herpes Simplex Virus)-DNA. The influence of CMV-reactivation on the endpoints was analysed by Cox proportional-hazard regression analysis. Time-dependency was evaluated by landmark analysis. RESULTS: Six out 97 died and five were discharged from the hospital within 72 hours and were excluded of the analysis. CMV reactivation occurred in 35 of the 86 (40.69%) analysed patients. HSV infection occurred in 23 of the 35 (65.7%) CMV reactivators. In 10 patients CMV-plasma-DNAemia appeared with a DNA-content below 600 copies/ml in four cases and a peak amount of 2,830 copies/ml on average. In patients with and without CMV reactivation mortality rates were similar (37.1% vs. 35.3%, P = 0.861), respectively. However, in the multivariate COX regression analyses CMV reactivation was independently associated with increased length of stay in the ICU (30.0, interquartile range 14 to 48 vs. 12.0, interquartile range 7 to 19 days; HR (hazard ratio) 3.365; 95% CI (confidence interval) 1.233 to 9.183, P = 0.018) and in the hospital (33.0, interquartile range 24 to 62 vs. 16.0, interquartile range 10 to 24 days, HR 3.3, 95% CI 1.78 to 6.25, P < 0.001) as well as prolonged mechanical ventilation (22.0, interquartile range 6 to 36 vs. 7.5, interquartile range 5 to 15.5 days; HR 2.6,CI 95% 1.39 to 4.94; P < 0.001) and impaired pulmonary gas exchange (six days, interquartile range 1 to 17, vs. three, interquartile range 1 to 7, days in reactivators vs. non-reactivators, P = 0.038). HSV reactivation proved not to be a risk factor for these adverse effects. CONCLUSIONS: These data indicate an independent correlation between CMV reactivation and increased morbidity in the well-defined group of nonimmunosuppressed patients with severe sepsis, but CMV reactivation had no impact on mortality in this group with low CMV-DNA plasma levels. Thus, the potential harms and benefits of antiviral treatment have to be weighed cautiously in patients with severe sepsis or septic shock.

Netrin-1 dampens pulmonary inflammation during acute lung injury.

RATIONALE: Acute lung injury (ALI) is an inflammatory disorder characterized by hypoxemia and diffuse infiltration of neutrophils into the alveolar space. The migration and extravasation of neutrophils is guided through positive guidance cues, such as chemokines. Recent work has identified the neuronal guidance protein netrin-1 to be a negative guidance cue for leukocyte migration and to hold antiinflammatory potential. OBJECTIVES: To test the role of pulmonary netrin-1 during ALI. METHODS: Pulmonary netrin-1 expression was evaluated during acute inflammation in vitro and in vivo; the netrin-1 promoter was studied using pGL4 luciferase reporter. ALI was induced through LPS inhalation and mechanical ventilation in wild-type, Ntn1(+/-), and A2BAR(-/-) animals. Exogenous netrin-1 was used to evaluate its impact on pulmonary inflammation. MEASUREMENTS AND MAIN RESULTS: Wild-type animals demonstrated repression of pulmonary netrin-1 after LPS inhalation. In vitro studies confirmed the repression of netrin-1. Studies in the putative netrin-1 promoter identified a nuclear factor-kappaB-dependent mechanism to be involved in this repression. Ntn1(+/-) animals demonstrated increased inflammatory changes after LPS inhalation compared with Ntn1(+/+) animals. Reconstitution with netrin-1 dampened the infiltration of neutrophils and cytokine production in the alveolar space. This effect was dependent on the adenosine 2b receptor. The importance of netrin-1 for the control of pulmonary inflammation could be corroborated in a model of ventilator-induced lung injury. CONCLUSIONS: Pulmonary netrin-1 levels are repressed during ALI. This results in pronounced pulmonary damage, an increased infiltration of neutrophils, and increased pulmonary inflammation. Exogenous netrin-1 significantly dampens the extent of ALI through the adenosine 2B receptor.

Local anesthetic effects on human neutrophil priming and activation.

BACKGROUND: The anti-inflammatory effects of local anesthetics (LAs) are well documented. Local anesthetics in micromolar concentrations inhibit extracellular oxygen release in isolated neutrophils; the underlying mechanism seems to be an inhibition of leukocyte priming. It remains unclear, however, if first, these effects also can be observed in whole blood, and second, if the priming of other neutrophil functions is similarly attenuated by LAs. Furthermore, the effects of LAs on intracellular generation of oxidative species remain to be investigated. METHODS: Whole-blood samples from healthy volunteers were incubated for 0, 1, or 3 hrs with different concentrations (10 to 10 M) of either lidocaine, ropivacaine, QX314, or NaCl 0.9% as control. Dihydroethidium was added to quantify oxidative burst. Samples were primed with platelet-activating factor (PAF, 10 M) and/or activated with formyl-methyl-leucyl-phenylalanine (10 M) for 15 mins each. After staining for CD11b and lysis of erythrocytes, samples were analyzed by flow cytometry. RESULTS: Priming of leukocytes is a relevant mechanism in whole blood. Platelet-activating factor stimulates the priming of oxidative burst and CD11b expression. Lidocaine up to millimolar concentrations did not affect the PAF priming and formyl-methyl-leucyl-phenylalanine activation of oxidative burst. The priming of CD11b expression and the priming and activation of changes in cell morphology were significantly attenuated by lidocaine. CONCLUSIONS: The intracellular generation of reactive oxygen species remains largely unaffected by LAs in clinical concentrations. This suggests that the anti-inflammatory effects of LAs do not interfere with the host defense.

Effects of 1400W and/or nitroglycerin on renal oxygenation and kidney function during endotoxaemia in anaesthetized rats.

1. The pathogenesis of acute renal failure (ARF) in sepsis is multifactorial. The role of nitric oxide (NO) in septic ARF has been a source of controversy. We hypothesized that endotoxaemia-induced exacerbation of inducible nitric oxide synthase (iNOS)-related NO release impairs renal oxygenation and contributes to ARF in anaesthetized rats. 2. In the present study, rats received lipopolysaccharide (2.5 mg/kg) for 30 min. Two hours later, fluid resuscitation was started (HES130; 5 mL/kg per h after a 5 mL/kg bolus) supplemented either by the NO donor nitroglycerin (NTG; 0.5 µg/kg per min after a 2 µg/kg bolus), the selective iNOS inhibitor 1400 W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po2 (µPo(2)) were recorded continuously. Furthermore, creatinine clearance, plasma NO(x) (nitrate + nitrite + S-nitrosothiols) levels and the expression of iNOS mRNA were measured. 3. Endotoxaemia reduced renal blood flow, decreased mean arterial pressure, resulted in anuria and was associated with an increase in plasma NO(x) levels and renal iNOS expression. Renal µPo2 deteriorated gradually during endotoxaemia and there was a significant decrease in renal O(2) delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo(2) or creatinine clearance over standard fluid resuscitation. The application of 1400 W+NTG significantly reduced plasma NO(x) levels compared with fluid resuscitation and NTG alone. 4. Neither iNOS inhibition, NO donation nor a combination of both showed beneficial effects on systemic haemodynamics, renal oxygenation and renal function compared with fluid resuscitation alone. Our results question the proposed key role of NO in the pathogenesis of septic ARF in rats.

Iloprost preserves renal oxygenation and restores kidney function in endotoxemia-related acute renal failure in the rat.

OBJECTIVE: To investigate that exogenous prostacyclin would counterbalance an endotoxemia-induced intrarenal vasoconstriction and would therefore have beneficial effects on kidney function. DESIGN: Prospective, randomized, controlled study. SETTING: University medical center research laboratory. SUBJECTS: Eighteen male Wistar rats. INTERVENTIONS: In anesthetized and ventilated animals, arterial blood pressure (mean arterial blood pressure [MAP]) and renal blood flow (RBF) were recorded. Renal microvascular Po2 (muPo2) and renal venous Po2 were continuously measured by phosphorescence lifetime technique. All animals received a 30-minute infusion of lipopolysaccharide (LPS) (2.5 mg/kg) to induce endotoxemia. One group of rats was not resuscitated. A second group received fluid resuscitation 90 minutes after stop of LPS infusion. In a third group of rats, the prostacyclin analogue iloprost (100 ng/kg/min) was continuously infused in addition to fluid resuscitation. Furthermore, in all the animals, plasma NOx levels, renal inducible nitric-oxide synthase (iNOS) messenger RNA (mRNA) expression, and creatinine clearance were determined. MEASUREMENTS AND MAIN RESULTS: During LPS infusion, MAP and RBF progressively dropped to 50% of baseline at 120 minutes. After an initial increase in MAP and RBF, start of fluid resuscitation with iloprost resulted in the stabilization of both parameters. All animals became anuric during endotoxemia. Only in animals receiving iloprost was creatinine clearance totally restored at the end of the experiment. Iloprost had no significant effects on average muPo2, but prevented the occurrence of cortical microcirculatory hypoxic areas. NOx levels and iNOS mRNA expression were significantly increased in all animals receiving LPS after 5 hours. There was no difference in NOx concentration between the different groups. In animals receiving iloprost, iNOS mRNA expression was significantly suppressed in the inner medulla. CONCLUSIONS: Iloprost significantly restored kidney function of endotoxemic rats to baseline values. This beneficial effect of iloprost on renal function might be addressed to an improvement in intrarenal oxygenation.

Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia.

The neuronal guidance molecule netrin-1 is linked to the coordination of inflammatory responses. Given that mucosal surfaces are particularly prone to hypoxia-elicited inflammation, we sought to determine the function of netrin-1 in hypoxia-induced inflammation. We detected hypoxia-inducible factor 1alpha (HIF-1alpha)-dependent induction of expression of the gene encoding netrin-1 (Ntn1) in hypoxic epithelia. Neutrophil transepithelial migration studies showed that by engaging A2B adenosine receptor (A2BAR) on neutrophils, netrin-1 attenuated neutrophil transmigration. Exogenous netrin-1 suppressed hypoxia-elicited inflammation in wild-type but not in A2BAR-deficient mice, and inflammatory hypoxia was enhanced in Ntn1(+/-) mice relative to that in Ntn1(+/+) mice. Our studies demonstrate that HIF-1alpha-dependent induction of netrin-1 attenuates hypoxia-elicited inflammation at mucosal surfaces.

Low-dose dexamethasone-supplemented fluid resuscitation reverses endotoxin-induced acute renal failure and prevents cortical microvascular hypoxia.

There is growing evidence that impairment in intrarenal oxygenation and hypoxic injury might contribute to the pathogenesis of septic renal failure. An important molecule known to act on the renal microvascular tone and therefore consequently being involved in the regulation of intrarenal oxygen supply is NO. The main production of NO under septic conditions derives from iNOS, an enzyme that can be blocked by dexamethasone (DEX). In an animal model of endotoxin-induced renal failure, we tested the hypothesis that inhibition of iNOS by low-dose DEX would improve an impaired intrarenal oxygenation and kidney function. Twenty-two male Wistar rats received a 30-min intravenous infusion of LPS (2.5 mg/kg) and consecutively developed endotoxemic shock. Two hours later, in 12 animals, fluid resuscitation was initiated. Six rats did not receive resuscitation; four animals served as time control. In addition to the fluid, six animals received a bolus of low-dose DEX (0.1 mg/kg). In these animals, the renal iNOS mRNA expression was significantly suppressed 3 h later. Dexamethasone prevented the appearance of cortical microcirculatory hypoxic areas, improved renal oxygen delivery, and significantly restored oxygen consumption. Besides a significant increase in MAP and renal blood flow, DEX restored kidney function and tubular sodium reabsorption to baseline values. In conclusion, treatment with low-dose DEX in addition to fluid resuscitation reversed endotoxin-induced renal failure associated by an improvement in intrarenal microvascular oxygenation. Therefore, low-dose DEX might have potential application in the prevention of septic acute renal failure.

[The intensive care in the German Diagnosis Related Groups-System]. / Die Intensivmedizin im deutschen DRG (Diagnosis Related Groups)-System.

The introduction of a prospective per case reimbursement system in the year 2004 in Germany put the high end intensive care medicine to a considerable financial risk on the basis of risk selection and high maintenance costs. To face this situation, the German Interdisciplinary Union for Intensive Care Medicine (DIVI) suggested to encode intensive care services as a DRG procedure and thus to make them relevant for reimbursement. That is the case now since 2007. The suggested procedure, called " complex intensive care treatment ", is based on a new intensive care scoring system ("Cost Predictor Score") which should quantify the costs per case. This paper provides an introduction into the GR-DRG (German Refined--Diagnosis Related Groups) health care reimbursement system and its financial implications for expensive hospital departments and presents a study carried out in the Anaesthesiological Intensive Care Unit of the Tübingen University Hospital examining the suitability of this new intensive care scoring system.

The inhibition of human neutrophil phagocytosis and oxidative burst by tricyclic antidepressants.

BACKGROUND: Tricyclic antidepressants are being investigated as long-acting analgesics for topical application in wounds or IV for postoperative pain relief. However, it remains unclear if tricyclic antidepressants affect the host defense and if reported toxic effects on neutrophils are of relevance in this setting. We therefore investigated the effects of amitriptyline, nortriptyline, and fluoxetine on human neutrophil phagocytosis, oxidative burst, and neutrophil toxicity in a human whole blood model. METHODS: Heparinized blood samples from healthy volunteers were incubated with amitriptyline, nortriptyline, or fluoxetine (10(-6) to 10(-3) M) for 0, 1, or 3 h. Staphylococcus aureus in a bacteria:neutrophil ratio of 5:1 and dihydroethidium (for the determination of oxidative burst) were added. Phagocytosis was stopped after 5, 10, 20, and 40 min. After lysis of red blood cells, samples were analyzed by flow cytometry. RESULTS: In concentrations up to 10(-4) M, none of the compounds affected neutrophil phagocytosis and oxidative burst. At 10(-3) M, all three compounds were highly toxic for neutrophils. Amitriptyline preserved morphological integrity, but completely suppressed neutrophil function. Nortriptyline and fluoxetine caused a marked disruption of neutrophils. The effects of the investigated antidepressants were not time-dependent. CONCLUSIONS: Phagocytosis and intracellular host defense are largely unaffected by antidepressants in concentrations of 10(-4) M and below. Our results confirm that antidepressants are highly toxic to neutrophils in millimolar concentrations. The neurotoxic effects and clinical side effects, but not effects on neutrophil functions, therefore, are likely to be the limiting factors in using antidepressants as analgesics.