Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs—when gently isolated—can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible)

Paralytic Impact of Centrifugation on Human Neutrophils

Centrifugation is a common step in most of the popular protocols for the isolation of neutrophils from whole blood. Inconsistent results from previous studies on neutrophils may originate from an underestimation of the centrifugation effect, as in consequence impaired, not native cells, being investigated. We hypothesize, that centrifugation significantly impairs major neutrophil functions. However, there is no data yet whether the application of g-force itself or the product of g-force and duration of centrifugation (=“g-time”) defines the impact on neutrophils. Neutrophils were isolated from whole blood via centrifugation with different g-times and subsequently analyzed via live cell imaging for migration, as well as via flow cytometry for oxidative burst and surface antigen expression. Chemotactic migration was significantly reduced with increasing g-time. Oxidative burst decreased likewise the higher the g-time applied. Expression of CD11b was no longer upregulated in response to an n-formylmethionine-leucyl-phenylalanine (fMLP) stimulus in neutrophils having experienced high g-time during the isolation process. We conclude that centrifugation “paralyzes” neutrophils in the form of a significant decrease in functionality. Future investigations on neutrophil granulocytes should reduce the g-time load as far as possible

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

Anaesthesia-related complications and side-effects in TAVI: a retrospective study in Germany

Objectives This study was performed to analyse anaesthesia-related complications and side effects in patients undergoing transcatheter aortic valve implantation (TAVI) under general anaesthesia. Design Retrospective study. Setting The study was performed as a single-centre study in a hospital of tertiary care in Germany. Participants All 853 patients, who underwent TAVI at the Universitatsklinikum Regensburg between January 2009 and July 2015, were included. 52.5% were female patients. Primary and secondary outcome measures We gathered information, such as recent illness, vital parameters and medication administered during the intervention and postoperatively for 12 hours. We analysed all anaesthesia-related complications and anaesthesia-related side effects that occurred during the intervention and entire hospital stay. Results We analysed all 853 TAVI procedures. The mean patient age was 79 +/- 6 years. In 99.5% of cases, we used volatile-based anaesthesia. 2.8% (n=24; transfemoral (TF): n=19 [3.8%]; transapical (TA): n=5 [1.4%]) of all cases suffered from anaesthesia-related complications. 819 (TF: n=447; TA: n=372) anaesthesia-related side effects occurred in 586 (68.7%, TF: n=325 [64.2%], TA: n=261 [75.2%]) patients. Neither the complications nor the side effects had any serious consequences. Intraoperative hypothermia in 44% of cases (TF: n=202 [39.9%]; TA: n=173 [49.9%]) and postoperative nausea and vomiting in 27% (n=232; TF: n=131 [25.9%], TA: n=101 [29.1%]) of cases were the most common anaesthesia-related side effects. Conclusion In this study, serious anaesthesia-related complications were rarely seen, and non-critical anaesthesia-related side effects could have been avoided through consistent prophylaxis and management. Therefore, despite their high anaesthetic risk, general anaesthesia is justifiable in patients who underwent TAVI

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing

Background: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. Methods: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. Results: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm−1s−1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [−0.1; 0.03], p = 0.387). Conclusions: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB

Das Ländliche als kulturelle Kategorie: aktuelle kulturwissenschaftliche Perspektiven auf Stadt-Land-Beziehungen

Das Ländliche hat Konjunktur. Zwischen medialer »Landlust« und realem Strukturwandel in den ländlichen Räumen Europas differenziert es sich dynamisch aus. Idylle oder Problemregion? Arbeitsort oder Freizeitpark? Repräsentationen und lebensweltliche Erfahrungen des Ländlichen avancieren zu einem alltagskulturellen Konfliktfeld, auf dem sich vor einem historischen Hintergrund elementare Problemlagen der Gegenwart und die Komplexität von kulturellen Land-Stadt-Beziehungen spiegeln.Die Beiträge des Bandes liefern hierzu Einsichten aus der Forschungsperspektive der Europäischen Ethnologie und rahmen das Thema damit für historische und gegenwartsbezogene Kulturanalysen begrifflich, methodisch und theoretisch

Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs—when gently isolated—can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible)

Perforation of myocardial wall and great vessels after cardiovascular interventions—a 5-year analysis

Background: Less invasive procedures have replaced open surgical treatment in many cardiovascular disorders. During these interventions, iatrogenic cardiac perforation may ensue, which is a severe complication and requires immediate diagnostic assessment and treatment. Methods: From March 2011 to April 2016, all patients referred to the Dept. of Cardio-thoracic Surgery with the diagnosis of iatrogenic perforation of myocardial wall or great vessels were included into the retrospective study. Complications during transapical transcutaneous aortic valve replacements (TAVR) procedures and percutaneous coronary intervention (PCI) were excluded from analysis. Symptoms, therapeutic strategy, intraoperative findings, and outcome were evaluated. Results: Forty-four patients suffered from myocardial wall or vessel perforation. Most common site of perforation were right (n=26; 59.1%) and left (n=8; 18.2%) ventricle. Other structures were involved in ten cases (22.7%). Open surgical treatment was required in 27 cases (61.4%). Mortality after left and right ventricular laceration was 75.0% and 11.5%, respectively. Most common cause of death was cardiocirculatory failure (n=5). Conclusions: Iatrogenic perforation of myocardial wall or central vessels during percutaneous interventional procedures is a rare but life-threatening complication. Despite immediate treatment efforts, mortality is high, particularly after left ventricular laceration

Complications after transcatheter aortic valve implantation using transfemoral and transapical approach in general anaesthesia.

Transcatheter aortic valve implantation (TAVI) is a minimally invasive procedure used to treat degenerative heart valve disease. The implantation requires a highly specific and interdisciplinary management approach. Currently, TAVI is performed with the patient under local or general anaesthesia.This study was a retrospective analysis of all TAVI procedures performed at the University Hospital of Regensburg between January 2009 and July 2015. All pre-, intra and postoperative data focusing on perioperative complications were recorded.A total of 853 transfemoral- and transapical-TAVI patients were included in the study. All patients underwent general anaesthesia. The ASA classifications were primarily 3-4. The average logistic EuroScores for the transfemoral- and transapical-TAVI patients were 18 ± 12% and 21 ± 15% (p = 0.002), respectively. The anaesthesia coverage time was 170 ± 49 min., including 37 ± 12 minutes for anaesthetic management. Overall, 458 complications were recorded; with pneumonia, acute renal failure, indication for a permanent pacemaker and non-extubation in the operating theatre the most frequently recorded complications.In the present study, we showed that our patients' outcomes are comparable to those reported in the available literature. Compared to TF, TA patients show an overall worse physical condition as well as a higher perioperative morbidity and mortality. Consequently TA patients need additional care and should only be operated in appropriately experienced medical centres