The Effect of Targeted Hyperoxemia on Brain Immunohistochemistry after Long-Term, Resuscitated Porcine Acute Subdural Hematoma and Hemorrhagic Shock.

Epidemiological data suggest that moderate hyperoxemia may be associated with an improved outcome after traumatic brain injury. In a prospective, randomized investigation of long-term, resuscitated acute subdural hematoma plus hemorrhagic shock (ASDH + HS) in 14 adult, human-sized pigs, targeted hyperoxemia (200 < PaO2 < 250 mmHg vs. normoxemia 80 < PaO2 < 120 mmHg) coincided with improved neurological function. Since brain perfusion, oxygenation and metabolism did not differ, this post hoc study analyzed the available material for the effects of targeted hyperoxemia on cerebral tissue markers of oxidative/nitrosative stress (nitrotyrosine expression), blood-brain barrier integrity (extravascular albumin accumulation) and fluid homeostasis (oxytocin, its receptor and the H2S-producing enzymes cystathionine-ß-synthase and cystathionine-γ-lyase). After 2 h of ASDH + HS (0.1 mL/kgBW autologous blood injected into the subdural space and passive removal of 30% of the blood volume), animals were resuscitated for up to 53 h by re-transfusion of shed blood, noradrenaline infusion to maintain cerebral perfusion pressure at baseline levels and hyper-/normoxemia during the first 24 h. Immediate postmortem, bi-hemispheric (i.e., blood-injected and contra-lateral) prefrontal cortex specimens from the base of the sulci underwent immunohistochemistry (% positive tissue staining) analysis of oxidative/nitrosative stress, blood-brain barrier integrity and fluid homeostasis. None of these tissue markers explained any differences in hyperoxemia-related neurological function. Likewise, hyperoxemia exerted no deleterious effects.

Functional and Morphological Differences of Muscle Mitochondria in Chronic Fatigue Syndrome and Post-COVID Syndrome.

Patients suffering from chronic fatigue syndrome (CFS) or post-COVID syndrome (PCS) exhibit a reduced physiological performance capability. Impaired mitochondrial function and morphology may play a pivotal role. Thus, we aimed to measure the muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity and assess mitochondrial morphology in CFS and PCS patients in comparison to healthy controls (HCs). Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers using high-resolution respirometry. Mitochondrial morphology (subsarcolemmal/intermyofibrillar mitochondrial form/cristae/diameter/circumference/area) and content (number and proportion/cell) were assessed via electron microscopy. Analyses included differences in OXPHOS between HC, CFS, and PCS, whereas comparisons in morphology/content were made for CFS vs. PCS. OXPHOS capacity of complex I, which was reduced in PCS compared to HC. While the subsarcolemmal area, volume/cell, diameter, and perimeter were higher in PCS vs. CFS, no difference was observed for these variables in intermyofibrillar mitochondria. Both the intermyofibrillar and subsarcolemmal cristae integrity was higher in PCS compared to CFS. Both CFS and PCS exhibit increased fatigue and impaired mitochondrial function, but the progressed pathological morphological changes in CFS suggest structural changes due to prolonged inactivity or unknown molecular causes. Instead, the significantly lower complex I activity in PCS suggests probably direct virus-induced alterations.

Relation of Plasma Catecholamine Concentrations and Myocardial Mitochondrial Respiratory Activity in Anesthetized and Mechanically Ventilated, Cardiovascular Healthy Swine.

Chronic heart failure is associated with reduced myocardial ß-adrenergic receptor expression and mitochondrial function. Since these data coincide with increased plasma catecholamine levels, we investigated the relation between myocardial ß-receptor expression and mitochondrial respiratory activity under conditions of physiological catecholamine concentrations. This post hoc analysis used material of a prospective randomized, controlled study on 12 sexually mature (age 20-24 weeks) Early Life Stress or control pigs (weaning at day 21 and 28-35 after birth, respectively) of either sex. Measurements in anesthetized, mechanically ventilated, and instrumented animals comprised serum catecholamine (liquid-chromatography/tandem-mass-spectrometry) and 8-isoprostane levels, whole blood superoxide anion concentrations (electron spin resonance), oxidative DNA strand breaks (tail moment in the "comet assay"), post mortem cardiac tissue mitochondrial respiration, and immunohistochemistry (ß2-adrenoreceptor, mitochondrial respiration complex, and nitrotyrosine expression). Catecholamine concentrations were inversely related to myocardial mitochondrial respiratory activity and ß2-adrenoceptor expression, whereas there was no relation to mitochondrial respiratory complex expression. Except for a significant, direct, non-linear relation between DNA damage and noradrenaline levels, catecholamine concentrations were unrelated to markers of oxidative stress. The present study suggests that physiological variations of the plasma catecholamine concentrations, e.g., due to physical and/or psychological stress, may affect cardiac ß2-adrenoceptor expression and mitochondrial respiration.

Porcine blood cell and brain tissue energy metabolism: Effects of "early life stress".

Background: Early Life Stress (ELS) may exert long-lasting biological effects, e.g., on PBMC energy metabolism and mitochondrial respiration. Data on its effect on brain tissue mitochondrial respiration is scarce, and it is unclear whether blood cell mitochondrial activity mirrors that of brain tissue. This study investigated blood immune cell and brain tissue mitochondrial respiratory activity in a porcine ELS model. Methods: This prospective randomized, controlled, animal investigation comprised 12 German Large White swine of either sex, which were weaned at PND (postnatal day) 28-35 (control) or PND21 (ELS). At 20-24 weeks, animals were anesthetized, mechanically ventilated and surgically instrumented. We determined serum hormone, cytokine, and "brain injury marker" levels, superoxide anion (O2 •¯) formation and mitochondrial respiration in isolated immune cells and immediate post mortem frontal cortex brain tissue. Results: ELS animals presented with higher glucose levels, lower mean arterial pressure. Most determined serum factors did not differ. In male controls, TNFα and IL-10 levels were both higher than in female controls as well as, no matter the gender in ELS animals. MAP-2, GFAP, and NSE were also higher in male controls than in the other three groups. Neither PBMC routine respiration and brain tissue oxidative phosphorylation nor maximal electron transfer capacity in the uncoupled state (ETC) showed any difference between ELS and controls. There was no significant relation between brain tissue and PBMC, ETC, or brain tissue, ETC, and PBMC bioenergetic health index. Whole blood O2 •¯ concentrations and PBMC O2 •¯ production were comparable between groups. However, granulocyte O2 •¯ production after stimulation with E. coli was lower in the ELS group, and this effect was sex-specific: increased O2 •¯ production increased upon stimulation in all control animals, which was abolished in the female ELS swine. Conclusion: This study provides evidence that ELS i) may, gender-specifically, affect the immune response to general anesthesia as well as O2 •¯ radical production at sexual maturity, ii) has limited effects on brain and peripheral blood immune cell mitochondrial respiratory activity, and iii) mitochondrial respiratory activity of peripheral blood immune cells and brain tissue do not correlate.

An exploratory study investigating the effect of targeted hyperoxemia in a randomized controlled trial in a long-term resuscitated model of combined acute subdural hematoma and hemorrhagic shock in cardiovascular healthy pigs.

Severe physical injuries and associated traumatic brain injury and/or hemorrhagic shock (HS) remain leading causes of death worldwide, aggravated by accompanying extensive inflammation. Retrospective clinical data indicated an association between mild hyperoxemia and improved survival and outcome. However, corresponding prospective clinical data, including long-term resuscutation, are scarce. Therefore, the present study explored the effect of mild hyperoxemia for 24 hours in a prospective randomized controlled trial in a long-term resuscitated model of combined acute subdural hematoma (ASDH) and HS. ASDH was induced by injecting 0.1 ml × kg-1 autologous blood into the subdural space and HS was triggered by passive removal of blood. After 2 hours, the animals received full resuscitation, including retransfusion of the shed blood and vasopressor support. During the first 24 hours, the animals underwent targeted hyperoxemia (PaO2 = 200 - 250 mmHg) or normoxemia (PaO2 = 80 - 120 mmHg) with a total observation period of 55 hours after the initiation of ASDH and HS. Survival, cardiocirculatory stability, and demand for vasopressor support were comparable between both groups. Likewise, humoral markers of brain injury and systemic inflammation were similar. Multimodal brain monitoring, including microdialysis and partial pressure of O2 in brain tissue, did not show significant differences either, despite a significantly better outcome regarding the modified Glasgow Coma Scale 24 hours after shock that favors hyperoxemia. In summary, the present study reports no deleterious and few beneficial effects of mild targeted hyperoxemia in a clinically relevant model of ASDH and HS with long-term resuscitation in otherwise healthy pigs. Further beneficial effects on neurological function were probably missed due to the high mortality in both experimental groups. The present study remains exploratory due to the unavailability of an a priori power calculation resulting from the lack of necessary data.

The effect of sodium thiosulfate on immune cell metabolism during porcine hemorrhage and resuscitation.

Introduction: Sodium thiosulfate (Na2S2O3), an H2S releasing agent, was shown to be organ-protective in experimental hemorrhage. Systemic inflammation activates immune cells, which in turn show cell type-specific metabolic plasticity with modifications of mitochondrial respiratory activity. Since H2S can dose-dependently stimulate or inhibit mitochondrial respiration, we investigated the effect of Na2S2O3 on immune cell metabolism in a blinded, randomized, controlled, long-term, porcine model of hemorrhage and resuscitation. For this purpose, we developed a Bayesian sampling-based model for 13C isotope metabolic flux analysis (MFA) utilizing 1,2-13C2-labeled glucose, 13C6-labeled glucose, and 13C5-labeled glutamine tracers. Methods: After 3 h of hemorrhage, anesthetized and surgically instrumented swine underwent resuscitation up to a maximum of 68 h. At 2 h of shock, animals randomly received vehicle or Na2S2O3 (25 mg/kg/h for 2 h, thereafter 100 mg/kg/h until 24 h after shock). At three time points (prior to shock, 24 h post shock and 64 h post shock) peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from whole blood, and cells were investigated regarding mitochondrial oxygen consumption (high resolution respirometry), reactive oxygen species production (electron spin resonance) and fluxes within the metabolic network (stable isotope-based MFA). Results: PBMCs showed significantly higher mitochondrial O2 uptake and lower O 2 • - production in comparison to granulocytes. We found that in response to Na2S2O3 administration, PBMCs but not granulocytes had an increased mitochondrial oxygen consumption combined with a transient reduction of the citrate synthase flux and an increase of acetyl-CoA channeled into other compartments, e.g., for lipid biogenesis. Conclusion: In a porcine model of hemorrhage and resuscitation, Na2S2O3 administration led to increased mitochondrial oxygen consumption combined with stimulation of lipid biogenesis in PBMCs. In contrast, granulocytes remained unaffected. Granulocytes, on the other hand, remained unaffected. O 2 • - concentration in whole blood remained constant during shock and resuscitation, indicating a sufficient anti-oxidative capacity. Overall, our MFA model seems to be is a promising approach for investigating immunometabolism; especially when combined with complementary methods.

13C-Metabolic flux analysis detected a hyperoxemia-induced reduction of tricarboxylic acid cycle metabolism in granulocytes during two models of porcine acute subdural hematoma and hemorrhagic shock.

Introduction: Supplementation with increased inspired oxygen fractions has been suggested to alleviate the harmful effects of tissue hypoxia during hemorrhagic shock (HS) and traumatic brain injury. However, the utility of therapeutic hyperoxia in critical care is disputed to this day as controversial evidence is available regarding its efficacy. Furthermore, in contrast to its hypoxic counterpart, the effect of hyperoxia on the metabolism of circulating immune cells remains ambiguous. Both stimulating and detrimental effects are possible; the former by providing necessary oxygen supply, the latter by generation of excessive amounts of reactive oxygen species (ROS). To uncover the potential impact of increased oxygen fractions on circulating immune cells during intensive care, we have performed a 13C-metabolic flux analysis (MFA) on PBMCs and granulocytes isolated from two long-term, resuscitated models of combined acute subdural hematoma (ASDH) and HS in pigs with and without cardiovascular comorbidity. Methods: Swine underwent resuscitation after 2 h of ASDH and HS up to a maximum of 48 h after HS. Animals received normoxemia (PaO2 = 80 - 120 mmHg) or targeted hyperoxemia (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation, thereafter PaO2 as in the control group). Blood was drawn at time points T1 = after instrumentation, T2 = 24 h post ASDH and HS, and T3 = 48 h post ASDH and HS. PBMCs and granulocytes were isolated from whole blood to perform electron spin resonance spectroscopy, high resolution respirometry and 13C-MFA. For the latter, we utilized a parallel tracer approach with 1,2-13C2 glucose, U-13C glucose, and U-13C glutamine, which covered essential pathways of glucose and glutamine metabolism and supplied redundant data for robust Bayesian estimation. Gas chromatography-mass spectrometry further provided multiple fragments of metabolites which yielded additional labeling information. We obtained precise estimations of the fluxes, their joint credibility intervals, and their relations, and characterized common metabolic patterns with principal component analysis (PCA). Results: 13C-MFA indicated a hyperoxia-mediated reduction in tricarboxylic acid (TCA) cycle activity in circulating granulocytes which encompassed fluxes of glutamine uptake, TCA cycle, and oxaloacetate/aspartate supply for biosynthetic processes. We further detected elevated superoxide levels in the swine strain characterized by a hypercholesterolemic phenotype. PCA revealed cell type-specific behavioral patterns of metabolic adaptation in response to ASDH and HS that acted irrespective of swine strains or treatment group. Conclusion: In a model of resuscitated porcine ASDH and HS, we saw that ventilation with increased inspiratory O2 concentrations (PaO2 = 200 - 250 mmHg for 24 h after treatment initiation) did not impact mitochondrial respiration of PBMCs or granulocytes. However, Bayesian 13C-MFA results indicated a reduction in TCA cycle activity in granulocytes compared to cells exposed to normoxemia in the same time period. This change in metabolism did not seem to affect granulocytes' ability to perform phagocytosis or produce superoxide radicals.

Cigarette smoke exposure reduces hemorrhagic shock induced circulatory dysfunction in mice with attenuated glucocorticoid receptor function.

Introduction: We previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS. Methods: After 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid-base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range. Results: Nor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals. Conclusion: Whether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

The effect of targeted hyperoxemia in a randomized controlled trial employing a long-term resuscitated, model of combined acute subdural hematoma and hemorrhagic shock in swine with coronary artery disease: An exploratory, hypothesis-generating study.

Controversial evidence is available regarding suitable targets for the arterial O2 tension (PaO2) after traumatic brain injury and/or hemorrhagic shock (HS). We previously demonstrated that hyperoxia during resuscitation from hemorrhagic shock attenuated cardiac injury and renal dysfunction in swine with coronary artery disease. Therefore, this study investigated the impact of targeted hyperoxemia in a long-term, resuscitated model of combined acute subdural hematoma (ASDH)-induced brain injury and HS. The prospective randomized, controlled, resuscitated animal investigation consisted of 15 adult pigs. Combined ASDH plus HS was induced by injection of 0.1 ml/kg autologous blood into the subdural space followed by controlled passive removal of blood. Two hours later, resuscitation was initiated comprising re-transfusion of shed blood, fluids, continuous i.v. noradrenaline, and either hyperoxemia (target PaO2 200 - 250 mmHg) or normoxemia (target PaO2 80 - 120 mmHg) during the first 24 h of the total of 54 h of intensive care. Systemic hemodynamics, intracranial and cerebral perfusion pressures, parameters of brain microdialysis and blood biomarkers of brain injury did not significantly differ between the two groups. According to the experimental protocol, PaO2 was significantly higher in the hyperoxemia group at the end of the intervention period, i.e., at 24 h of resuscitation, which coincided with a higher brain tissue PO2. The latter persisted until the end of observation period. While neurological function as assessed using the veterinary Modified Glasgow Coma Score progressively deteriorated in the control group, it remained unaffected in the hyperoxemia animals, however, without significant intergroup difference. Survival times did not significantly differ in the hyperoxemia and control groups either. Despite being associated with higher brain tissue PO2 levels, which were sustained beyond the intervention period, targeted hyperoxemia exerted neither significantly beneficial nor deleterious effects after combined ASDH and HS in swine with pre-existing coronary artery disease. The unavailability of a power calculation and, thus, the limited number of animals included, are the limitations of the study.

The H2S Donor Sodium Thiosulfate (Na2S2O3) Does Not Improve Inflammation and Organ Damage After Hemorrhagic Shock in Cardiovascular Healthy Swine.

We previously demonstrated marked lung-protective properties of the H2S donor sodium thiosulfate (Na2S2O3, STS) in a blinded, randomized, controlled, long-term, resuscitated porcine model of swine with coronary artery disease, i.e., with decreased expression of the H2S-producing enzyme cystathionine-γ-lyase (CSE). We confirmed these beneficial effects of STS by attenuation of lung, liver and kidney injury in mice with genetic CSE deletion (CSE-ko) undergoing trauma-and-hemorrhage and subsequent intensive care-based resuscitation. However, we had previously also shown that any possible efficacy of a therapeutic intervention in shock states depends both on the severity of shock as well as on the presence or absence of chronic underlying co-morbidity. Therefore, this prospective, randomized, controlled, blinded experimental study investigated the effects of the STS in cardiovascular healthy swine. After anesthesia and surgical instrumentation, 17 adult Bretoncelles-Meishan-Willebrand pigs were subjected to 3 hours of hemorrhage by removal of 30% of the blood volume and titration of the mean arterial pressure (MAP) ≈ 40 ± 5 mmHg. Afterwards, the animals received standardized resuscitation including re-transfusion of shed blood, fluids, and, if needed, continuous i.v. noradrenaline to maintain MAP at pre-shock values. Animals were randomly allocated to either receive Na2S2O3 or vehicle control starting 2 hours after initiation of shock until 24 hours of resuscitation. The administration of Na2S2O3 did not alter survival during the observation period of 68 hours after the initiation of shock. No differences in cardio-circulatory functions were noted despite a significantly higher cardiac output, which coincided with significantly more pronounced lactic acidosis at 24 hours of resuscitation in the Na2S2O3 group. Parameters of liver, lung, and kidney function and injury were similar in both groups. However, urine output was significantly higher in the Na2S2O3 group at 24 hours of treatment. Taken together, this study reports no beneficial effect of Na2S2O3 in a clinically relevant model of hemorrhagic shock-and-resuscitation in animals without underlying chronic cardiovascular co-morbidity.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine-γ-Lyase Knockout Mice With Diabetes Type 1.

Background: Sodium thiosulfate (STS) is a recognized drug with antioxidant and H2S releasing properties. We recently showed that STS attenuated organ dysfunction and injury during resuscitation from trauma-and-hemorrhage in CSE-ko mice, confirming its previously described organ-protective and anti-inflammatory properties. The role of H2S in diabetes mellitus type 1 (DMT1) is controversial: genetic DMT1 impairs H2S biosynthesis, which has been referred to contribute to endothelial dysfunction and cardiomyopathy. In contrast, development and severity of hyperglycemia in streptozotocin(STZ)-induced DMT1 was attenuated in CSE-ko mice. Therefore, we tested the hypothesis whether STS would also exert organ-protective effects in CSE-ko mice with STZ-induced DMT1, similar to our findings in animals without underlying co-morbidity. Methods: Under short-term anesthesia with sevoflurane and analgesia with buprenorphine CSE-ko mice underwent DMT1-induction by single STZ injection (100 µg⋅g-1). Seven days later, animals underwent blast wave-induced blunt chest trauma and surgical instrumentation followed by 1 h of hemorrhagic shock (MAP 35 ± 5 mmHg). Resuscitation comprised re-transfusion of shed blood, lung-protective mechanical ventilation, fluid resuscitation and continuous i.v. norepinephrine together with either i.v. STS (0.45 mg⋅g-1) or vehicle (n = 9 in each group). Lung mechanics, hemodynamics, gas exchange, acid-base status, stable isotope-based metabolism, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, chemokines, and immunoblotting. Results: Diabetes mellitus type 1 was associated with more severe circulatory shock when compared to our previous study using the same experimental design in CSE-ko mice without co-morbidity. STS did not exert any beneficial therapeutic effect. Most of the parameters measured of the inflammatory response nor the tissue expression of marker proteins of the stress response were affected either. Conclusion: In contrast to our previous findings in CSE-ko mice without underlying co-morbidity, STS did not exert any beneficial therapeutic effect in mice with STZ-induced DMT1, possibly due to DMT1-related more severe circulatory shock. This result highlights the translational importance of both integrating standard ICU procedures and investigating underlying co-morbidity in animal models of shock research.

H2S in Critical Illness-A New Horizon for Sodium Thiosulfate?

Ever since the discovery of endogenous H2S and the identification of its cytoprotective properties, efforts have been made to develop strategies to use H2S as a therapeutic agent. The ability of H2S to regulate vascular tone, inflammation, oxidative stress, and apoptosis might be particularly useful in the therapeutic management of critical illness. However, neither the inhalation of gaseous H2S, nor the administration of inorganic H2S-releasing salts or slow-releasing H2S-donors are feasible for clinical use. Na2S2O3 is a clinically approved compound with a good safety profile and is able to release H2S, in particular under hypoxic conditions. Pre-clinical studies show promise for Na2S2O3 in the acute management of critical illness. A current clinical trial is investigating the therapeutic potential for Na2S2O3 in myocardial infarct. Pre-eclampsia and COVID-19 pneumonia might be relevant targets for future clinical trials.

Systemic calcitonin gene-related peptide receptor antagonism decreases survival in a porcine model of polymicrobial sepsis: blinded randomised controlled trial.

BACKGROUND: Calcitonin gene-related peptide (CGRP) and procalcitonin, which are overexpressed in sepsis, exert distinct immunomodulatory effects mediated through the CGRP receptor. The CGRP receptor antagonist olcegepant improves survival in murine sepsis. This study evaluated whether CGRP receptor antagonism is similarly beneficial in a porcine model of polymicrobial sepsis. METHODS: We conducted a prospective randomised, controlled, investigator-blinded trial in adult pigs of either sex, that were anaesthetised and ventilated before sepsis was induced by polymicrobial (autologous) faecal peritonitis. After the onset of early septic shock (systolic blood pressure <90 mm Hg or >10% decline from baseline MAP), pigs were resuscitated (i.v. fluid/antibiotics/vasopressors) and randomised to receive either i.v. olcegepant (n=8) or vehicle control (n=8). The primary outcome was time to death, euthanasia required up to 72 h after surgery (according to predefined severe cardiorespiratory failure), or both. Secondary outcomes included haemodynamic changes, and systemic as well as organ inflammation (mRNA expression). RESULTS: Septic shock developed 8.7 h (inter-quartile range, 5.8-11.1 h) after the onset of faecal peritonitis. Olcegepant worsened survival, with 6/8 pigs randomised to the control group surviving 72.0 h (50.9-72.0 h), compared with 3/8 pigs receiving olcegepant surviving 51.3 h (12.5-72.0 h; P=0.01). At 48 h, lower MAP and higher cardiac output occurred in pigs receiving olcegepant. Cardiac, hepatic, and renal injury was not different between pigs randomised to receive olcegepant or vehicle. Olcegepant reduced mRNA expression of several inflammation-related cytokines and CD68+ macrophages in liver but not in lung tissue. CONCLUSIONS: CGRP receptor antagonism with olcegepant was not beneficial in this porcine model of polymicrobial sepsis, which closely mimics human sepsis.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine Gamma Lyase (CSE) Knockout Mice.

BACKGROUND: Sodium thiosulfate (Na2S2O3) is a clinically established drug with antioxidant and sulphide-releasing properties. Na2S2O3 mediated neuro- and cardioprotective effects in ischemia/reperfusion models and anti-inflammatory effects in LPS-induced acute lung injury. Moreover, Na2S2O3 improved lung function during resuscitation from hemorrhagic shock in swine with pre-existing atherosclerosis, characterized by decreased expression of cystathionine γ-lyase (CSE), a major source of hydrogen sulfide (H2S) synthesis in the vasculature. Based on these findings, we investigated the effects of Na2S2O3 administration during resuscitation from trauma-and-hemorrhage in mice under conditions of whole body CSE deficit. METHODS: After blast wave-induced blunt chest trauma and surgical instrumentation, CSE knockout (CSE-/-) mice underwent 1 h of hemorrhagic shock (MAP 35 ±â€Š5 mm Hg). At the beginning of resuscitation comprising retransfusion, norepinephrine support and lung-protective mechanical ventilation, animals received either i.v. Na2S2O3 (0.45 mg g-1, n = 12) or vehicle (saline, n = 13). Hemodynamics, acid-base status, metabolism using stable isotopes, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, mitochondrial respiratory capacity, and immunoblotting. RESULTS: Na2S2O3 treatment improved arterial paO2 (P = 0.03) coinciding with higher lung tissue glucocorticoid receptor expression. Norepinephrine requirements were lower in the Na2S2O3 group (P < 0.05), which was associated with lower endogenous glucose production and higher urine output. Na2S2O3 significantly increased renal tissue IκBα and heme oxygenase-1 expression, whereas it lowered kidney IL-6 and MCP-1 levels. CONCLUSION: Na2S2O3 exerted beneficial effects during resuscitation of murine trauma-and-hemorrhage in CSE-/- mice, confirming and extending the previously described organ-protective and anti-inflammatory properties of Na2S2O3. The findings make Na2S2O3 a potentially promising therapeutic option in the context of impaired CSE activity and/or reduced endogenous H2S availability.

NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer's Disease-Related Proteins.

Alzheimer's disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aß) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aß-related proteins on the single-cell level, we could verify the important role of intracellular Aß-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

Target arterial PO2 according to the underlying pathology: a mini-review of the available data in mechanically ventilated patients.

There is an ongoing discussion whether hyperoxia, i.e. ventilation with high inspiratory O2 concentrations (FIO2), and the consecutive hyperoxaemia, i.e. supraphysiological arterial O2 tensions (PaO2), have a place during the acute management of circulatory shock. This concept is based on experimental evidence that hyperoxaemia may contribute to the compensation of the imbalance between O2 supply and requirements. However, despite still being common practice, its use is limited due to possible oxygen toxicity resulting from the increased formation of reactive oxygen species (ROS) limits, especially under conditions of ischaemia/reperfusion. Several studies have reported that there is a U-shaped relation between PaO2 and mortality/morbidity in ICU patients. Interestingly, these mostly retrospective studies found that the lowest mortality coincided with PaO2 ~ 150 mmHg during the first 24 h of ICU stay, i.e. supraphysiological PaO2 levels. Most of the recent large-scale retrospective analyses studied general ICU populations, but there are major differences according to the underlying pathology studied as well as whether medical or surgical patients are concerned. Therefore, as far as possible from the data reported, we focus on the need of mechanical ventilation as well as the distinction between the absence or presence of circulatory shock. There seems to be no ideal target PaO2 except for avoiding prolonged exposure (> 24 h) to either hypoxaemia (PaO2 < 55-60 mmHg) or supraphysiological (PaO2 > 100 mmHg). Moreover, the need for mechanical ventilation, absence or presence of circulatory shock and/or the aetiology of tissue dysoxia, i.e. whether it is mainly due to impaired macro- and/or microcirculatory O2 transport and/or disturbed cellular O2 utilization, may determine whether any degree of hyperoxaemia causes deleterious side effects.

ΔMST and the Regulation of Cardiac CSE and OTR Expression in Trauma and Hemorrhage.

Genetic deletion of 3-mercaptopyruvate sulfurtransferase (MST) is known to result in hypertension and cardiac hypertrophy in older mice, and is associated with increased anxiety-like behaviors. Endogenous hydrogen sulfide (H2S) produced by MST in the mitochondria is also known to be involved in physiological and cellular bioenergetics, and its dysfunction associated with depressive behavior and increased cardiovascular morbidity. Interestingly, early life stress has been shown to lead to a significant loss of cystathionine-γ-lyase (CSE) and oxytocin receptor (OTR) expression in the heart. Thus, we were interested in testing the hypothesis of whether genetic MST mutation (ΔMST) would affect cardiac CSE and OTR expression and affect the mitochondrial respiration in a clinically relevant, resuscitated, mouse model of trauma and hemorrhagic shock. In ΔMST mice, we found a reduction of CSE and OTR in both the naive as well as injured state, in contrast to the wild type (wt) controls. Interestingly, the ΔMST showed a different complex IV response to injury than the wt controls, although our claims are based on the non-demonstrated assumption that naive wt and naive ΔMST mice have comparable complex IV activity. Finally, hemorrhagic shock led to a reduction of CSE and OTR, confirming previous results in the injured mouse heart. To date, the exact mechanisms of the cardiac interaction between H2S and OT are not clear, but they point the way to potential cardioprotective therapies.

Effects of Acute Subdural Hematoma-Induced Brain Injury On Energy Metabolism in Peripheral Blood Mononuclear Cells.

ABSTRACT: In activated immune cells, differentiation and function are determined by cell type-specific modifications of metabolic patterns. After traumatic brain injury both immune cell activation and suppression were reported. Therefore, we sought to explore immune cell energy metabolism in a long-term, resuscitated porcine model of acute subdural hematoma (ASDH)-induced acute brain injury devoid of impaired systemic hemodynamics and oxygen transport.Before and up to 50 h after induction of ASDH, peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation, and cell metabolism was analyzed using high-resolution respirometry for mitochondrial respiration and electron spin resonance for reactive oxygen species production. After incubation with stable isotope-labeled 1,2-13C2-glucose or 13C5-glutamine, distinct labeling patterns of intermediates of glycolysis or tricarboxylic acid (TCA) cycle and 13CO2 production were measured by gas chromatography-mass spectroscopy. Principal component analysis was followed by a varimax rotation on the covariance across all measured variables and all measured time points.After ASDH induction, average PBMC metabolic activity remained unaffected, possibly because strict adherence to intensive care unit guidelines limited trauma to ASDH induction without any change in parameters of systemic hemodynamics, oxygen transport, and whole-body metabolism. Despite decreased glycolytic activity fueling the TCA cycle, the principal component analysis indicated a cell type-specific activation pattern with biosynthetic and proliferative characteristics.

Corticotropin-releasing hormone (CRH) alters mitochondrial morphology and function by activating the NF-kB-DRP1 axis in hippocampal neurons.

Neuronal stress-adaptation combines multiple molecular responses. We have previously reported that thorax trauma induces a transient loss of hippocampal excitatory synapses mediated by the local release of the stress-related hormone corticotropin-releasing hormone (CRH). Since a physiological synaptic activity relies also on mitochondrial functionality, we investigated the direct involvement of mitochondria in the (mal)-adaptive changes induced by the activation of neuronal CRH receptors 1 (CRHR1). We observed, in vivo and in vitro, a significant shift of mitochondrial dynamics towards fission, which correlated with increased swollen mitochondria and aberrant cristae. These morphological changes, which are associated with increased NF-kB activity and nitric oxide concentrations, correlated with a pronounced reduction of mitochondrial activity. However, ATP availability was unaltered, suggesting that neurons maintain a physiological energy metabolism to preserve them from apoptosis under CRH exposure. Our findings demonstrate that stress-induced CRHR1 activation leads to strong, but reversible, modifications of mitochondrial dynamics and morphology. These alterations are accompanied by bioenergetic defects and the reduction of neuronal activity, which are linked to increased intracellular oxidative stress, and to the activation of the NF-kB/c-Abl/DRP1 axis.