Role of mitochondrial DNA level in epidural-related maternal fever: a single-centre, observational, pilot study.

INTRODUCTION: Epidural analgesia has been associated with intrapartum maternal fever development. Epidural-related maternal fever (ERMF) is believed to be based on a non-infectious inflammatory reaction. Circulating cell-free mitochondrial deoxyribonucleic acid (mtDNA) is one of the possible triggers of sterile inflammatory processes; however, a connection has not been investigated so far. Therefore, this study aimed to investigate cell-free mtDNA alterations in women in labour with ERMF in comparison with non-febrile women. MATERIAL AND METHODS: A total of 60 women in labour were assessed for maternal temperature every 4 h and blood samples were obtained at the beginning and after delivery. Depending on the analgesia and the development of fever (axillary temperature ≥ 37.5 °C), the women were allocated either to the group of no epidural analgesia (n = 17), to epidural analgesia no fever (n = 34) or to ERMF (n = 9). Circulating cell-free mtDNA was analysed in the maternal plasma for the primary outcome whereas secondary outcomes include the evaluation of inflammatory cytokine release, as well as placental inflammatory signs. RESULTS: Of the women with epidural analgesia, 20% (n = 9) developed ERMF and demonstrated a decrease of circulating mtDNA levels during labour (p = 0.04), but a trend towards higher free nuclear DNA. Furthermore, women with maternal pyrexia showed a 1.5 fold increased level of Interleukin-6 during labour. A correlation was found between premature rupture of membranes and ERMF. CONCLUSIONS: The pilot trial revealed an evident obstetric anaesthesia phenomenon of maternal fever due to epidural analgesia in 20% of women in labour, demonstrating counterregulated free mtDNA and nDNA. Further work is urgently required to understand the connections between the ERMF occurrence and circulating cell-free mtDNA as a potential source of sterile inflammation. TRIAL REGISTRATION: NCT0405223 on clinicaltrials.gov (registered on 25/07/2019).

Argon Preconditioning Protects Airway Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Stress.

BACKGROUND: Oxidative stress is the predominant pathogenic mechanism of ischaemia-reperfusion (IR) injury. The noble gas argon has been shown to alleviate oxidative stress-related myocardial and cerebral injury. The risk of lung IR injury is increased in some major surgeries, reducing clinical outcome. However, no study has examined the lung-protective efficacy of argon preconditioning. The present study investigated the protective effects of argon preconditioning on airway epithelial cells exposed to hydrogen peroxide (H2O2) to induce oxidative stress. METHODS: A549 airway epithelial cells were treated with a cytotoxic concentration of H2O2 after exposure to standard air or 30 or 50% argon/21% oxygen/5% carbon dioxide/rest nitrogen for 30, 45 or 180 min. Cells were stained with annexin V/propidium iodide, and apoptosis was evaluated by fluorescence-activated cell sorting. Protective signalling pathways activated by argon exposure were identified by Western blot analysis for phosphorylated candidate molecules of the mitogen-activated protein kinase and protein kinase B (Akt) pathways. RESULTS: Preconditioning with 50% argon for 30, 45 and 180 min and 30% argon for 180 min caused significant protection of A549 cells against H2O2-induced apoptosis, with increases in cellular viability of 5-47% (p < 0.0001). A small adverse effect was also observed, which presented as a 12-15% increase in cellular necrosis in argon-treated groups. Argon exposure resulted in early activation of c-Jun N-terminal kinase (JNK) and p38, peaking 10- 30 min after the start of preconditioning, and delayed activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, peaking after 60-90 min. CONCLUSIONS: Argon preconditioning protects airway epithelial cells from H2O2-induced apoptotic cell death. Argon activates the JNK, p38, and ERK1/2 pathways, but not the Akt pathway. The cytoprotective properties of argon suggest possible prophylactic applications in surgery-related IR injury of the lungs.

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition.

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4ß2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the ß3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.

Brief High Oxygen Concentration Induces Oxidative Stress in Leukocytes and Platelets: A Randomized Cross-over Pilot Study in Healthy Male Volunteers.

BACKGROUND: Supplemental oxygen is administered routinely in the clinical setting to relieve or prevent tissue hypoxia, but excessive exposure may induce oxidative damage or disrupt essential homeostatic functions. It is speculated that oxidative stress in leukocytes and platelets may contribute to vascular diseases by promoting inflammation and cell aggregation. METHODS: In this pilot study 30 healthy male volunteers (18-65 years) were exposed to high oxygen concentration (non-rebreather mask, 8 L/min, 100% O2) and synthetic air (non-rebreather mask, 8 L/min, 21% O2) in a cross-over design for 20 min at a 3-week interval. Venous blood samples were obtained at baseline and 1, 3, and 6 h postintervention. Primary outcome was generation of reactive oxygen species in leukocytes as measured by the redox-sensitive fluorescent dye dihydrorhodamine 123. Additional outcomes were oxidative stress in platelets and platelet aggregation as measured by thromboelastography (ROTEM) and Multiplate analyses. FINDINGS: High oxygen exposure induced oxidative stress in leukocytes as evidenced by significantly higher mean fluorescence intensity (MFI) compared with synthetic air at 3 h postintervention (47% higher, P = 0.015) and 6 h postintervention (37% higher, P = 0.133). Oxidative stress was also detectable in platelets (33% higher MFI in comparison with synthetic air at 6 h, P = 0.024; MFI 20% above baseline at 3 h, P  = 0.036; 37% above baseline at 6 h, P = 0.002). ROTEM analyses demonstrated reduced mean clotting time 1 h postintervention compared with baseline (-4%, P = 0.049), whereas there were no significant effects on other surrogate coagulation parameters. CONCLUSION: Clinically relevant oxygen exposure induces oxidative stress in leukocytes and platelets, which may influence the immune and clotting functions of these cells.

Remote ischemic perconditioning attenuates adverse cardiac remodeling and preserves left ventricular function in a rat model of reperfused myocardial infarction.

AIMS: Remote ischemic conditioning (RIC) is considered a potential clinical approach to reduce myocardial infarct size and ameliorate adverse post-infarct left ventricular (LV) remodeling, however the mechanisms are unknown. The aim was to clarify the impact of RIC on Neuregulin-1 (NRG-1)/ErbBs expression, inflammation and LV hemodynamic function. METHODS AND RESULTS: Male Sprague-Dawley rats were subjected to 30 min occlusion of the left coronary artery (LCA) followed by 2 weeks of reperfusion and separated into three groups: (1) sham operated (without LCA occlusion); (2) Myocardial ischemia/reperfusion (MIR) and (3) remote ischemic perconditioning group (MIR + RIPerc). Cardiac structural and functional changes were evaluated by echocardiography and on the isolated working heart system. The level of H3K4me3 at the NRG-1 promoter, and both plasma and LV tissue levels of NRG-1 were assessed. The expression of pro-inflammatory cytokines, ECM components and ErbB receptors were assessed by RT-qPCR. MIR resulted in a significant decrease in LV function and enlargement of LV chamber. This was accompanied with a decrease in the level of H3K4me3 at the NRG-1 promoter. Consequently NRG-1 protein levels were reduced in the infarcted myocardium. Subsequently, an upregulated influx of CD68+ macrophages, high expression of MMP-2 and -9 as well as an increase of IL-1ß, TLR-4, TNF-α, TNC expression were observed. In contrast, RIPerc significantly decreased inflammation and improved LV function in association with the enhancement of NRG-1 levels and ErbB3 expression. CONCLUSIONS: These findings may reveal a novel anti-remodeling and anti-inflammatory effect of RIPerc, involving activation of NRG-1/ErbB3 signaling.

Argon preconditioning enhances postischaemic cardiac functional recovery following cardioplegic arrest and global cold ischaemia.

OBJECTIVES: Previous studies demonstrated that preconditioning with argon gas provided a marked reduction in inflammation and apoptosis and increased myocardial contractility in the setting of acute myocardial ischaemia-reperfusion (IR). There is substantial evidence that myocardial IR injury following cardioplegic arrest is associated with the enhancement of apoptosis and inflammation, which is considered to play a role in cardiac functional impairment. Therefore, the present study was designed to clarify whether preconditioning with argon gas enhances recovery of cardiac function following cardioplegic arrest. METHODS: Sprague-Dawley rats were anaesthetized and ventilated and allocated to (i) the control group (control IR, n = 10) and (ii) the in vivo group (argon IR), which received 3 cycles of argon (50% argon, 21% oxygen and 29% nitrogen, n = 10) administered for 5 min interspersed with 5 min of a gas mixture (79% nitrogen and 21% oxygen). The hearts were excised and then evaluated in an erythrocyte-perfused isolated working heart system. Cold ischaemia (4°C) for 60 min was induced by histidine-tryptophan-ketoglutarate cardioplegia, followed by 40 min of reperfusion. Cardiac functional parameters were assessed. In left ventricular tissue samples, the expressions of extracellular-regulated kinase (ERK1/2), AKT serine/threonine kinase (Akt), jun N-terminal kinase (JNK), endothelial nitric oxide synthase (eNOS) and HMGB1: high-mobility group box 1 (HMGB1) protein were assessed by western blot, and high-energy phosphates were evaluated by high-performance liquid chromatography. RESULTS: At the end of reperfusion, the rats preconditioned with argon showed significantly enhanced recovery of cardiac output (101 ± 6% vs 87 ± 11%; P < 0.01), stroke volume (94 ± 4% vs 80 ± 11%; P = 0.001), external heart work (100 ± 6% vs 81 ± 13%; P < 0.001) and coronary flow (90 ± 13% vs 125 ± 21%; P < 0.01) compared with the control IR group. These results were accompanied by a significant increase in the levels of myocardial phosphocreatine (23.71 ± 2.07 µmol/g protein vs the control IR group, 13.50 ± 4.75; P = 0.001) and maintained adenosine triphosphate levels (13.62 ±1.89 µmol/g protein vs control IR group adenosine triphosphate: 10.08 ± 1.94 µmol/g; P = 0.017). Additionally, preconditioning with argon markedly reduced the activation of JNK (0.11 ± 0.01 vs 0.25 ± 0.03; P = 0.005) and the expression of HMGB1 protein (0.52 ± 0.04 vs 1.5 ± 0.10; P < 0.001) following reperfusion. CONCLUSIONS: Preconditioning with argon enhanced cardiac functional recovery in rat hearts arrested with histidine-tryptophan-ketoglutarate cardioplegia, thereby representing a potential novel cardioprotective approach in cardiac surgery.

Hyperoxia Induces Inflammation and Cytotoxicity in Human Adult Cardiac Myocytes.

Supplemental oxygen (O2) is used as adjunct therapy in anesthesia, emergency, and intensive care medicine. We hypothesized that excessive O2 levels (hyperoxia) can directly injure human adult cardiac myocytes (HACMs). HACMs obtained from the explanted hearts of transplantation patients were exposed to constant hyperoxia (95% O2), intermittent hyperoxia (alternating 10 min exposures to 5% and 95% O2), constant normoxia (21% O2), or constant mild hypoxia (5% O2) using a bioreactor. Changes in cell morphology, viability as assessed by lactate dehydrogenase (LDH) release and trypan blue (TB) staining, and secretion of vascular endothelial growth factor (VEGF), macrophage migration inhibitory factor (MIF), and various pro-inflammatory cytokines (interleukin, IL; chemokine C-X-C motif ligand, CXC; granulocyte-colony stimulating factor, G-CSF; intercellular adhesion molecule, ICAM; chemokine C-C motif ligand, CCL) were compared among treatment groups at baseline (0 h) and after 8, 24, and 72 h of treatment. Changes in HACM protein expression were determined by quantitative proteomic analysis after 48 h of exposure. Compared with constant normoxia and mild hypoxia, constant hyperoxia resulted in a higher TB-positive cell count, greater release of LDH, and elevated secretion of VEGF, MIF, IL-1ß, IL-6, IL-8, CXCL-1, CXCL-10, G-CSF, ICAM-1, CCL-3, and CCL-5. Cellular inflammation and cytotoxicity gradually increased and was highest after 72 h of constant and intermittent hyperoxia. Quantitative proteomic analysis revealed that hypoxic and hyperoxic O2 exposure differently altered the expression levels of proteins involved in cell-cycle regulation, energy metabolism, and cell signaling. In conclusion, constant and intermittent hyperoxia induced inflammation and cytotoxicity in HACMs. Cell injury occurred earliest and was greatest after constant hyperoxia, but even relatively brief repeating hyperoxic episodes induced a substantial inflammatory response.