Propofol Ameliorates Exaggerated Human Neutrophil Activation in a LPS Sepsis Model.

BACKGROUND: Sepsis is a leading cause of morbidity and mortality worldwide. Many patients suffering from sepsis are treated on intensive care units and many of them require mechanical ventilation under sedation or general anesthesia. Propofol, a drug used for these purposes, is known to interact with polymorphonuclear granulocytes (PMNs). Therefore, the aim of this study was to investigate the influence of propofol on PMN functions after experimental Gram-negative induced sepsis using lipopolysaccharide (LPS) stimulation. METHODS: A total of 34 granulocyte-enriched samples were collected from healthy subjects. PMNs were isolated by density gradient centrifugation and incubated simultaneously with either 6 µg/mL or 60 µg/mL propofol, or none (control). Additionally, the experimental sepsis samples were incubated with either 40 pg/mL or 400 pg/mL LPS. Live cell imaging was conducted in order to observe granulocyte chemotactic migration, ROS production, and NETosis. Flow cytometry was used to analyze viability and antigen expression. RESULTS: Propofol led to significantly reduced PMN track length (p < 0.001) and track speed (p < 0.014) after LPS-induced sepsis in a dose-dependent manner. NETosis (p = 0.018) and ROS production (p = 0.039) were accelerated by propofol without LPS incubation, indicating improved immune function. Propofol also ameliorated LPS-induced increased NETosis and ROS-production. Antigen expression for CD11b, CD62l and CD66b was unaffected by propofol. CONCLUSION: Propofol improves LPS-induced exaggerated PMN activation in an ex vivo model. Beneficial effects due to restored immune function in septic patients might be possible, but needs further investigation.

Butyrylcholinesterase as a perioperative complication marker in patients after transcatheter aortic valve implantation: a prospective observational study.

OBJECTIVES: Transcatheter aortic valve implantation (TAVI) is performed in elderly patients with severe aortic valve stenosis and increased operative risks. We tested the hypothesis that acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) have a predictive value for prevalent complications after TAVI and could serve as indicators of systemic inflammation in the early postoperative period. DESIGN: Prospective observational study. SETTING: This study is a secondary analysis of multicentre CESARO- study. PARTICIPANTS: 48 patients with TAVI were included and 43 obtained the complete assessment. PRIMARY AND SECONDARY OUTCOME MEASURES: Patients' clinical parameters, demographic data, peripheral AChE and BChE activities and routine blood markers were assessed throughout the perioperative period using bedside point-of-care measurements for AChE and BChE. Postoperative complication screening was conducted up to the third postoperative day and included infections, delirium and heart-rhythm disturbances. After assessment, the patients were divided into complication and noncomplication group. RESULTS: Of 43 patients, 24 developed postsurgical complications (55.8%). Preoperative assessment showed no significant differences regarding demographic data and laboratory markers, but preoperative BChE levels were significantly lower in patients who developed postoperative complications (complication group 2589.2±556.4 vs noncomplication group 3295.7±628.0, Cohen's r=0.514, p<0.001). In complication group, we observed an early, sustained reduction in BChE activity from preoperative to postoperative period. In complication group, BChE levels were significantly lower at each time point compared with noncomplication group. AChE activity showed no significant difference between both groups. Complication group also had longer stay in hospital overall. CONCLUSION: BChE could be a useful perioperative biomarker to identify patients with a higher risk for postoperative complications after TAVI. By using point-of-care measurements, the levels of BChE are fast available and can lead to an early targeted therapy. Predicting the length of the hospital stay might play an important role in staff and resource management for these patients. TRIAL REGISTRATION NUMBER: NCT01964274; Post-results.

Remote and Anesthetic-Induced Myocardial Preconditioning Is Preserved in Atherosclerotic LDL Receptor-/- Mice In Vivo.

INTRODUCTION: In the animal model, preconditioning is a powerful weapon against ischemic damage. The reason why the human heart cannot be protected from ischemic damage by preconditioning remains unclear. There are assumptions that the lack of preconditioning in humans is caused by concomitant diseases such as dyslipoproteinemia and arteriosclerosis. This study investigates whether dyslipoproteinemia and the resulting arteriosclerosis can be a cause of a reduced precondition effect of heart in mice. METHODS: LDL receptor-deficient mice were fed a long-term (14-16 weeks) high-fat atherogenic diet to induce arteriosclerosis. Arteriosclerosis was identified by histological examination and vessel contraction tests. LDLR-/- and wild-type mice were randomly assigned to anesthetic-induced, remote ischemic, or no preconditioning. All mice were subjected to 45 minutes of coronary artery occlusion and 180 minutes of reperfusion. The area at risk and infarct size were determined by Evans Blue and triphenyltetrazolium chloride staining. RESULTS: Histopathological examination showed atherosclerosis in high-fat atherogenic fed LDLR-/- mice, and the vessel relaxation capacity was significantly reduced compared to wild-type mice. In the wild type, as expected, infarct size was significantly reduced by preconditioning compared to the control. In LDLR-/- mice, infarct size was significantly reduced by preconditioning compared to the control. Surprisingly, the LDLR-/- control group also had a significantly reduced infarct size compared to the wild-type control group. CONCLUSION: We were able to demonstrate that a high-fat diet morphologically and functionally triggered atherosclerosis in LDLR-/- mice. Interestingly, LDLR-/- mice with an atherogenic diet had smaller infarct sizes compared to wild-type mice. Moreover, preconditioning additionally reduced myocardial infarct size in LDLR-/- mice. A long-term high-fat atherogenic diet and preconditioning seem to result in additive cardioprotection in LDLR-/- mice.

Potential Impact of Local Anesthetics Inducing Granulocyte Arrest and Altering Immune Functions on Perioperative Outcome.

INTRODUCTION: Local anesthetics (LAs) are frequently used during anesthesia; however, they may influence granulocyte function which in turn could modify immune responses in the perioperative period. Therefore, the aim of this study was to investigate the impact of clinically used doses of bupivacaine and lidocaine on granulocyte function with regard to migration, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETosis) formation, and viability. METHODS: A total of 38 granulocyte-enriched samples from healthy subjects were obtained by whole blood lysis. Polymorphonuclear neutrophil (PMN) samples were incubated simultaneously with different concentrations of either bupivacaine (0.03-3.16 mmol/L) or lidocaine (0.007-14.21 mmol/L), or without drug (control). Live cell imaging was conducted in order to observe granulocyte chemotaxis, migration, ROS production, and NETosis. Flow cytometry was used to analyze viability and antigen expression. RESULTS: The track length (TL) of PMNs exposed to bupivacaine concentrations of 0.16 mmol/L and above significantly decreased compared to the control. Low concentrations of lidocaine were associated with slight but significant increases in TL, whereas this changed with concentrations above 1.4 mmol/L, showing a significant decrease in TL. PMN incubated with bupivacaine concentrations of 1.58 mmol/L and above or lidocaine concentrations of at least 3.6 mmol/L showed no migration or chemotaxis at all. Time to onset of maximal ROS production and time for half-maximal NETosis decreased in a dose-dependent manner for both substances. Equipotency in NETosis induction was reached by bupivacaine (1.1 mmol/L) at significantly lower concentrations than lidocaine (7.96 mmol/L). Cell viability and oxidative burst were unaffected by LAs. CONCLUSION: Local anesthetics in clinically used doses ameliorate granulocyte defense mechanisms, thus indicating their potentially decisive effect during the perioperative period.

Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging.

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn's disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders.

Facial and Oral Cross-Contamination of a 3-Year-Old Child With High Concentration Capsaicin: A Case Report.

High-concentration topical capsaicin is used to treat different neuropathic pain states. We present a case in which a 3-year-old child orally ingested capsaicin after touching her mother's arm that had been treated with a high-concentration capsaicin patch 3 hours earlier. The child suffered extreme pain and swelling of the lips and tongue. After the use of cleansing gel, external cooling, and drinking milk, the pain lessened over half an hour and subsided after 2 hours. This report aims to raise awareness for this formerly unreported mode of oral contamination.

Therapeutic Anticoagulation with Argatroban and Heparins Reduces Granulocyte Migration: Possible Impact on ECLS-Therapy?

INTRODUCTION: Anticoagulants such as argatroban and heparins (low-molecular-weight and unfractionated) play an immense role in preventing thromboembolic complications in clinical practice. Nevertheless, they can also have a negative effect on the immune system. This study is aimed at investigating the influence of these substances on polymorphonuclear neutrophils (PMNs), whose nonspecific defense mechanisms can promote thrombogenesis. METHODS: Blood samples from 30 healthy volunteers were investigated, whereby PMNs were isolated by density gradient centrifugation and incubated with 0.8 µg/mL of argatroban, 1.0 U/mL of low-molecular-weight heparin (LMWH), 1.0 U/mL of unfractionated heparin (UFH), or without drug (control). A collagen-cell mixture was prepared and filled into 3D µ-slide chemotaxis chambers (IBIDI® GmbH, Germany). Stimulation was initiated by using a chemokine gradient of n-formyl-methionine-leucyl-phenylalanine (fMLP), and microscopic observation was conducted for 4.5 hours. The cells' track length and track straightness, as well as the number of attracted granulocytes, level of ROS (reactive oxygen species) production, and NET (neutrophil extracellular traps) formation, were analyzed and categorized into migration distances and time periods. RESULTS: All three anticoagulants led to significantly reduced PMN track lengths, with UFH having the biggest impact. The number of tracks observed in the UFH group were significantly reduced compared to the control group. Additionally, the UFH group demonstrated a significantly lower track straightness compared to the control. ROS production and NET formation were unaffected. CONCLUSION: Our data provide evidence that anticoagulants have an inhibitory effect on the extent of PMN migration and chemotactic migration efficiency, thus indicating their potential immune-modulatory and prothrombotic effects.

Accumulations of von Willebrand factor within ECMO oxygenators: Potential indicator of coagulation abnormalities in critically ill patients?

Clot formation within membrane oxygenators (MOs) remains a critical problem during extracorporeal membrane oxygenation (ECMO). The composition of the clots-in particular, the presence of von Willebrand factor (vWF)-may be an indicator for prevalent nonphysiological flow conditions, foreign body reactions, or coagulation abnormalities in critically ill patients. Mats of interwoven gas exchange fibers from randomly collected MOs (PLS, Maquet, Rastatt, Germany) of 21 patients were stained with antibodies (anti-vWF and anti-P-selectin) and counterstained with 4',6-diamidino-2-phenylindole. The extent of vWF-loading was correlated with patient and technical data. While 12 MOs showed low vWF-loadings, 9 MOs showed high vWF-loading with highest accumulations close to crossing points of adjacent gas fibers. The presence and the extent of vWF-fibers/"cobwebs," leukocytes, platelet-leukocyte aggregates (PLAs), and P-selectin-positive platelet aggregates were independent of the extent of vWF-loading. However, the highly loaded MOs were obtained from patients with a significantly elevated SOFA score, severe thrombocytopenia, and persistent liver dysfunction. The coagulation abnormalities of these critically ill patients may cause an accumulation of the highly thrombogenic and elongated high-molecular-weight vWF multimers in the plasma which will be trapped in the MOs during the ECMO therapy.

Impact of hydroxyethyl starch and modified fluid gelatin on granulocyte phenotype and function.

BACKGROUND: Patients with neutropenia or granulocyte dysfunction may require granulocyte transfusions for adequate immune restoration. High-molecular-weight hydroxyethyl starch (HES) is the most commonly used sedimentation agent to enhance granulocyte collection efficiency. However, authorities recently restricted the use of HES due to its unfavorable risk-benefit profile. As modified fluid gelatin (MFG) is already used as an alternative sedimentation agent, we tested the hypothesis that MFG is not inferior to HES in terms of the functionality and viability of granulocytes. STUDY DESIGN AND METHODS: Granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15%, or 30% MFG (Gelafundin) or HES (Hespan), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ROS) production, neutrophil extracellular trap formation (NETosis), antigen expression, and viability were subsequently investigated in vitro. RESULTS: Relative to the controls, all three concentrations of HES compared to only 15% and 30% MFG lowered migration distances, and the 15% and 30% concentrations of both sedimentation agents reduced track straightness. HES resulted in lower CD11b expression and higher CD62L expression compared to MFG and the controls, whereas the differences for CD66b did not reach statistical significance. No significant differences in the timing of ROS production or NETosis, or in neutrophil viability or respiratory burst were observed. CONCLUSION: These results indicate that MFG is not inferior to HES in terms of granulocyte function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with HES.

Intravenous hydrogen sulfide does not induce neuroprotection after aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury in a human-like porcine model of ubiquitous arteriosclerosis.

OBJECTIVE: In rodents, intravenous sulfide protected against spinal cord ischemia/reperfusion (I/R) injury during aortic balloon occlusion. We investigated the effect of intravenous sulfide on aortic occlusion-induced porcine spinal cord I/R injury. METHODS: Anesthetized and mechanically ventilated "familial hypercholesterolemia Bretoncelles Meishan" (FBM) pigs with high-fat-diet-induced hypercholesterolemia and atherosclerosis were randomized to receive either intravenous sodium sulfide 2 h (initial bolus, 0.2 mg kg body weight (bw)-1; infusion, 2 mg kg bw-1 h-1; n = 4) or vehicle (sodium chloride, n = 4) prior to 45 min of thoracic aortic balloon occlusion and for 8 h during reperfusion (infusion, 1 mg kg bw-1 h-1). During reperfusion, noradrenaline was titrated to maintain blood pressure at above 80% of the baseline level. Spinal cord function was assessed by motor evoked potentials (MEPs) and lower limb reflexes using a modified Tarlov score. Spinal cord tissue damage was evaluated in tissue collected at the end of experiment using hematoxylin and eosin and Nissl staining. RESULTS: A balloon occlusion time of 45 min resulted in marked ischemic neuron damage (mean of 16% damaged motoneurons in the anterior horn of all thoracic motor neurons) in the spinal cord. In the vehicle group, only one animal recovered partial neuronal function with regain of MEPs and link motions at each time point after deflating. All other animals completely lost neuronal functions. The intravenous application of sodium sulfide did not prevent neuronal cell injury and did not confer to functional recovery. CONCLUSION: In a porcine model of I/R injury of the spinal cord, treatment with intravenous sodium sulfide had no protective effect in animals with a pre-existing arteriosclerosis.

Life span of different extracorporeal membrane systems for severe respiratory failure in the clinical practice.

Over the past decade, veno-venous extracorporeal membrane oxygenation (vvECMO) has been increasingly utilized in respiratory failure in patients. This study presents our institution´s experience focusing on the life span of ECMO systems reflecting the performance of a particular system. A retrospective review of our ECMO database identified 461 adult patients undergoing vvECMO (2010-2017). Patients that required more than one system and survived the first exchange >24 hours (n = 139) were included. Life span until the first exchange and exchange criteria were analyzed for all systems (PLS, Cardiohelp HLS-set, both Maquet Cardiopulmonary, Rastatt, Germany; Deltastream/Hilite7000LT, iLA-activve, Xenios/NovaLung, Heilbronn, Germany; ECC.O5, LivaNova, Mirandola, Italy). At our ECMO center, the frequency of a system exchange was 30%. The median (IQR) life span was 9 (6-12) days. There was no difference regarding the different systems (p = 0.145 and p = 0.108, respectively). However, the Deltastream systems were exchanged more frequently due to elective technical complications (e. g. worsened gas transfer, development of coagulation disorder, increased bleedings complications) compared to the other exchanged systems (p = 0.013). In summary, the used ECMO systems are safe and effective for acute respiratory failure. There is no evidence for the usage of a specific system. Only the increased predictability of an imminent exchange preferred the usage of a Deltastream system. However, the decision to use a particular system should not depend solely on the possible criteria for an exchange.

Leukocyte Adhesion as an Indicator of Oxygenator Thrombosis During Extracorporeal Membrane Oxygenation Therapy?

Thrombosis is the most common technical complication with extracorporeal membrane oxygenation (ECMO). Accumulations of leukocytes on the gas exchange membranes within a membrane oxygenator (MO) may initiate thrombosis and influence outcome. MOs (n = 41) were removed routinely from adult patients on ECMO, preserved, and analyzed for their cellular deposits using nuclear (4',6-diamidino-2-phenylindole) and cell type-specific markers (CD45; von Willebrand factor, vWF). The extent of cellular colonization was correlated with patient data. Blood contact caused adhesion of leukocytes and accumulation of vWF. Six MOs contained "pseudomembranes" (PM). MOs with PM were from younger patients (median [interquartile range {IQR}]; age, 36 [30-47] vs. 61 [51-71] years; p = 0.040) and the leukocyte count before ECMO was on average higher (21 [16-24] vs. 15 [8-18] ×10 per L; p = 0.051) compared with PM-free MOs. The development of PMs did not influence pressure drop across the MO. Data indicating coagulation disorder within the MOs (d-dimers, fibrinogen, and platelets) were not significantly different between the groups. There was only one acute MO thrombosis in a PM-free MO. The support time of the analyzed MOs with PM tended to be longer when compared with PM-free MOs (11 [6-19] vs. 8 [5-11] days). Nevertheless, all patients with MOs with PMs were successfully weaned (6/6 vs. 17/35) and discharged from hospital (6/6 vs. 17/35; p = 0.027) compared with patients with PM-free MOs. In conclusion, elderly people on ECMO showed reduced PM formation that may reduce the risk of MO thrombosis. Younger patients had no negative effect.