Boris Nohé

Influence of fluid resuscitation on renal microvascular PO2 in a normotensive rat model of endotoxemia

IntroductionSeptic renal failure is often seen in the intensive care unit but its pathogenesis is only partly understood. This study, performed in a normotensive rat model of endotoxemia, tests the hypotheses that endotoxemia impairs renal microvascular PO2 (μPO2) and oxygen consumption (VO2,ren), that endotoxemia is associated with a diminished kidney function, that fluid resuscitation can restore μPO2, VO2,ren and kidney function, and that colloids are more effective than crystalloids.MethodsMale Wistar rats received a one-hour intravenous infusion of lipopolysaccharide, followed by resuscitation with HES130/0.4 (Voluven®), HES200/0.5 (HES-STERIL®® 6%) or Ringers lactate. The renal μPO2 in the cortex and medulla and the renal venous PO2 were measured by a recently published phosphorescence lifetime technique.ResultsEndotoxemia induced a reduction in renal blood flow and anuria, while the renal μPO2 and VO2,ren remained relatively unchanged. Resuscitation restored renal blood flow, renal oxygen delivery and kidney function to baseline values, and was associated with oxygen redistribution showing different patterns for the different compounds used. HES200/0.5 and Ringers lactate increased the VO2,ren, in contrast to HES130/0.4.ConclusionThe loss of kidney function during endotoxemia could not be explained by an oxygen deficiency. Renal oxygen redistribution could for the first time be demonstrated during fluid resuscitation. HES130/0.4 had no influence on the VO2,ren and restored renal function with the least increase in the amount of renal work.

A Pilot Open-Label Study of the Efficacy of Subanesthetic Isomeric S( + )-Ketamine in Refractory CRPS Patients

OBJECTIVE Complex regional pain syndrome (CRPS) is a severe neuropathic pain state that is often disproportionate to the initial trauma. Associated features are autonomic dysregulation, swelling, motor dysfunction, and trophic changes to varying degrees. Despite a multitude of treatment modalities, a subgroup of CRPS patients remain refractory to all standard therapies. In these patients, the disease may spread extraterritorially, which results in severe disability. A critical involvement of N-methyl-D-aspartate receptors (NMDARs) has been demonstrated both clinically and by animal experimentation. NMDA antagonists may be effective in many neuropathic pain states. In long-standing, generalized CRPS, we investigated the effects of S(+)-ketamine on pain relief and somatosensory features, assessed by quantitative sensory testing (QST). METHODS Four refractory CRPS patients received continous S(+)-ketamine-infusions, gradually titrated (50 mg/day-500 mg/day) over a 10-day period. Pain intensities (average, peak, and least pain) and side effects were rated on visual analogue scales, during a 4-day baseline, over 10 treatment days, and 2 days following treatment. QST (thermo-, mechanical detection, and pain thresholds) was analyzed at baseline and following treatment. RESULTS Subanesthetic S(+)-ketamine showed no reduction of pain and effected no change in thermo- and mechanical detection or pain thresholds. This procedure caused no relevant side effects. The lack of therapeutic response in the first four patients led to termination of this pilot study. CONCLUSION S(+)-ketamine can be gradually titrated to large doses (500 mg/day) without clinically relevant side effects. There was no pain relief or change in QST measurements in this series of long-standing severe CRPS patients.

Synthetic colloids attenuate leukocyte-endothelial interactions by inhibition of integrin function

Background: It has been suspected that synthetic colloids may interfere with leukocyte adhesion by down-regulation of endothelial cell adhesion molecules. Although inhibition of endothelial inflammation might reduce leukocyte-related tissue injury, the same mechanism may be detrimental for host defense during severe infection. Regarding the widespread use of colloids, the authors performed a laboratory investigation to determine the mechanisms by which synthetic colloids interfere with leukocyte-endothelial interactions. Methods: Adhesion molecule expression on native and cytokine-activated endothelium from umbilical veins was measured after pretreatment with gelatin and various preparations of dextran or hydroxyethyl starch. Inhibition of neutrophil adhesion to activated endothelium was examined in a flow chamber by perfusion of untreated and colloid-treated neutrophils over colloid-pretreated endothelium at 2 dyn/cm2. Comparisons were made between untreated controls, colloid-pretreated endothelium, and colloid-cotreated neutrophils. Results: Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and P-selectin were not attenuated by any colloid. Accordingly, colloid pretreatment of endothelium alone did not reduce neutrophil adhesion. In contrast, when neutrophils were cotreated by addition of colloids to the perfusate immediately before perfusion, adhesion decreased by 31–51% (P < 0.05) regardless of the colloid type. As indicated by the twofold increased rolling fractions, this reduction was due to an inhibition of neutrophil integrins. Conclusions: This study shows that synthetic colloids inhibit neutrophil adhesion by a neutrophil-dependent mechanism rather than interfering with endothelial cell activation. This suggests that inhibition of leukocyte sequestration by volume support is a common and transient phenomenon depending on the colloid concentration in plasma.

Certain batches of albumin solutions influence the expression of endothelial cell adhesion molecules.

Objective: Increased levels of soluble adhesion molecules, a decreased PO2/FIO2 ratio and a tendency to worsened outcome have been reported following the use of human albumin in critical illness. The reasons are not yet understood. Since albumin solutions have previously been shown to contain proinflammatory mediators, a direct upregulation of adhesion molecules by contaminated batches may explain these findings. To examine this, we studied the effects of different albumin preparations on endothelial cell adhesion molecules in vitro. Design: Experimental study. Setting: Laboratory for cell biology. Methods: Human umbilical venous endothelial cell cultures (n = 4) were incubated for 6 h at 5 mg/ml with four different human albumin solutions (HA1–4) from different manufacturers. Medium served as the control. Using flow cytometry, the effects on E-selectin, ICAM-1 and VCAM-1 expression were determined on unstimulated cells and on cells stimulated with tumour necrosis factor α at 0.5 ng/ml for 4 h. Measurements and results: On unstimulated cells, HA1 and HA4, two different batches from the same manufacturer, increased ICAM-1 by 22 % and 15 %, respectively. After stimulation, both solutions resulted in a 19 % increased expression of E-Selectin. In addition, HA4 decreased VCAM-1 on stimulated cells (p≤ 0.05). Two albumin preparations from other manufacturers did not produce significant effects. Conclusions: Some albumin solutions directly modulate adhesion molecule expression on endothelial cells. This may, at least in part, explain the previous finding of increased soluble adhesion molecules and a decreased PO2/FIO2 ratio in critically ill patients undergoing volume replacement with human albumin.

Local anesthetics impair human granulocyte phagocytosis activity, oxidative burst, and CD11b expression in response to Staphylococcus aureus.

Background With invasion of bacteria, the host defense system is activated by a complex cascade of various mechanisms. Local anesthetics previously were shown to interact with diverse components of the immune response, such as leukocyte adherence on endothelial monolayers, oxidative burst, or crosstalk within lymphocyte subset populations. However, effects of newer local anesthetics like bupivacaine and ropivacaine on antibacterial host defense—primarily phagocytosis activity, oxidative burst, or CD11b expression—still remain unclear. Methods Whole blood samples were preincubated with local anesthetics (lidocaine, 9.2, 92.2, and 1,846 &mgr;m; bupivacaine, 6.1, 61, and 770 &mgr;m; ropivacaine, 6.4, 64, and 801 &mgr;m). For the oxidative burst and CD11b assay, dihydroethidium was added to the probes. After viable Staphylococcus aureus was added in a 5 to 1 ratio following leukocyte count, phagocytosis was stopped at different times, and staining with monoclonal antibodies was performed for subsequent flow cytometric analysis of phagocytosis activity, oxidative burst, and CD11b expression. Results Granulocyte phagocytosis activity, CD11b expression, and generation of reactive oxygen species were significantly reduced by lidocaine (P < 0.0002) and bupivacaine (P < 0.005) in the highest concentration (1,846 &mgr;m and 770 &mgr;m, respectively). The capability of granulocytes to ingest bacteria was significantly depressed only by lidocaine (P < 0.003). Ropivacaine had no significant effect on any parameter investigated. Conclusions Local anesthetic dose and structure dependently inhibit inflammatory and immunologic parameters of granulocyte functions. Ropivacaine shows low interference with granulocyte immunologic and inflammatory functions.

Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury

IntroductionThis study was carried out to determine whether interactions of cell activation, shear stress and platelets at sites of endothelial injury explain the paradoxical maldistribution of activated leukocytes during sepsis away from local sites of infection towards disseminated leukocyte accumulation at remote sites.MethodsHuman umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were activated with lipopolysaccharide at 100 and 10 ng/ml to achieve adhesion molecule patterns as have been reported from the hyper- and hypo-inflammatory stage of sepsis. To examine effects of leukocyte activation on leukocyte-endothelial interactions, activated HUVEC were perfused with activated and non-activated neutrophils in a parallel plate flow chamber. Adhesion molecule expression and function were assessed by flow cytometry and blocking antibodies. In a subset of experiments the sub-endothelial matrix was exposed and covered with platelets to account for the effects of endothelial injury. To investigate interactions of these effects with flow, all experiments were done at various shear stress levels (3 to 0.25 dyne/cm2). Leukocyte-endothelial interactions were analyzed by videomicroscopy and analysis of covariance.ResultsActivation of neutrophils rendered adhesion increasingly dependent on shear stress reduction. At normal shear stress, shedding of L-selectin decreased adhesion by 56%. Increased rolling fractions of activated PMN at low shear stress revealed impaired integrin affinity despite numerical up-regulation of CD11b. On sub-maximally activated, intact HUVEC shear stress became the prevailing determinant of adhesion. Presence of a platelet-covered injury with high surface density of P-selectin was the strongest variable for adhesion. When compared to maximally activated HUVEC, platelets increased neutrophil adhesion by 2.7-fold. At sub-maximal activation a 10-fold increase was observed (P < 0.05 for all).ConclusionsL-selectin shedding and integrin dysfunction render leukocyte adhesion increasingly susceptible to shear stress and alternative adhesion receptors. In combination, these effects inhibit recruitment to normally perfused sites with intact endothelium and favor maldistribution towards sites with compromised perfusion or endothelial injury.

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 1400W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po2 (µPo2) were recorded continuously. Furthermore, creatinine clearance, plasma NOx (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 NOx levels and renal iNOS expression. Renal µPo2 deteriorated gradually during endotoxaemia and there was a significant decrease in renal O2 delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo2 or creatinine clearance over standard fluid resuscitation. The application of 1400W+NTG significantly reduced plasma NOx 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.

ImageStream cytometry extends the analysis of phagocytosis and oxidative burst

Abstract 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.

Influence of underlying disease on the outcome of critically ill patients with acute renal failure

Background and objective: The development of acute renal failure (ARF) in critically ill patients is associated with an increase in hospital mortality. Recently, it was shown that starting renal replacement therapy early and using high-filtrate flow rates can improve the outcome, but this could not be confirmed in later investigations. Studying selected patient subgroups could provide a useful basis for patient selection in future trials evaluating the outcome of renal replacement therapies. We, therefore, investigated the impact of the underlying disease on the outcome of patients with ARF. Methods: We retrospectively analysed 306 patients with ARF who were treated with renal replacement therapy. Patients were classified according to six initial diagnosis groups: haemorrhagic shock, post-cardiac surgery, post-liver transplantation, trauma, severe sepsis and miscellaneous. Univariate and multivariate multiple logistic regression analysis was used to determine which factors influenced the outcome. Results: Underlying disease proved to be the only independent risk factor for mortality that was present at intensive care unit (ICU) admission (P = 0.047). Patients with severe sepsis had a significantly higher mortality rate (68%) than ARF patients as a whole (51%) (P = 0.02). Length of stay in the ICU, the use of catecholamines, the delay before ARF onset, and the correlation between APACHE II score and ICU length of stay proved to be additional independent predictors of outcome. Conclusions: Patient selection and subgroup definition according to the underlying disease could augment the usefulness of future trials evaluating the outcome of ARF.

Effects of magnetic cell separation on monocyte adhesion to endothelial cells under flow

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 °C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 °C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non‐activated monocytes are needed for adhesion assays under flow.