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.

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.

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.

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 (Na2S2O3) during resuscitation from hemorrhagic shock in swine with preexisting atherosclerosis.

Controversial data are available on hydrogen sulfide (H2S) during hemorrhage and resuscitation, depending on timing, dosing, mode of application, and the H2S donor used. Sodium thiosulfate (Na2S2O3) is a recognized drug devoid of major side effects, which attenuated murine acute lung injury and cerebral ischemia/reperfusion injury. Therefore, we tested the hypothesis whether Na2S2O3 would mitigate organ dysfunction in porcine hemorrhage-and-resuscitation. We studied animals with pre-existing coronary artery disease because of the reduced coronary arterial expression of the H2S producing enzyme cystathionine-γ-lyase (CSE) in this prospective, randomized, controlled, blinded experimental study. 20 anesthetized and instrumented pigs underwent 3 h of hemorrhage (removal of 30 % of the blood volume and subsequent titration of mean arterial pressure to 40 mmHg). Resuscitation (72 h) comprised re-transfusion of shed blood, crystalloids, and continuous i.v. norepinephrine. Animals randomly received vehicle or Na2S2O3 (0.1 g·kg-1 h-1) for 24 h. Before, at the end of and every 24 h after shock, hemodynamics, metabolism, blood gases, lung, heart, kidney, and liver function and injury were evaluated together with cytokines and parameters of oxidative and nitrosative stress. Immediate post mortem lung, kidney, heart, and liver specimen were analyzed for marker proteins of inflammation and oxidative and nitrosative stress and mitochondrial respiratory activity in the heart, kidney, and liver. Immuno-histochemical analysis comprised lung extra-vascular albumin accumulation, nitrotyrosine formation, and CSE and glucocorticoid receptor (GCR) expression. Na2S2O3 significantly attenuated shock-induced impairment of lung mechanics and gas exchange (plateau and positive end-expiratory pressure at 72 h p = 0.0006/p = 0.0264; Horovitz index at 48 h p = 0.0261), which coincided with a higher tissue GCR expression (p = 0.0415). During resuscitation from hemorrhagic shock Na2S2O3 attenuated shock-induced acute lung injury in co-morbid swine, most likely due to a GCR expression related mechanism.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging.

BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

Effects of Hyperoxia During Resuscitation From Hemorrhagic Shock in Swine With Preexisting Coronary Artery Disease.

OBJECTIVES: Investigation of the effects of hyperoxia during resuscitation from hemorrhagic shock in swine with preexisting coronary artery disease. DESIGN: Prospective, controlled, randomized trial. SETTING: University animal research laboratory. SUBJECTS: Nineteen hypercholesterolemic pigs with preexisting coronary artery disease. INTERVENTIONS: Anesthetized, mechanically ventilated, and surgically instrumented pigs underwent 3 hours of hemorrhagic shock (removal of 30% of the calculated blood volume and subsequent titration of mean arterial blood pressure ≈40 mm Hg). Postshock resuscitation (48 hr) comprised retransfusion of shed blood, crystalloids (balanced electrolyte solution), and norepinephrine support. Pigs were randomly assigned to "control" (FIO2 0.3, adjusted for arterial oxygen saturation ≥ 90%) and "hyperoxia" (FIO2 1.0 for 24 hr) groups. MEASUREMENTS AND MAIN RESULTS: Before, at the end of shock and every 12 hours of resuscitation, datasets comprising hemodynamics, calorimetry, blood gases, cytokines, and cardiac and renal function were recorded. Postmortem, organs were sampled for immunohistochemistry, western blotting, and mitochondrial high-resolution respirometry. Survival rates were 50% and 89% in the control and hyperoxia groups, respectively (p = 0.077). Apart from higher relaxation constant τ at 24 hours, hyperoxia did not affect cardiac function. However, troponin values were lower (2.2 [0.9-6.2] vs 6.9 [4.8-9.8] ng/mL; p < 0.05) at the end of the experiment. Furthermore, hyperoxia decreased cardiac 3-nitrotyrosine formation and increased inducible nitric oxide synthase expression. Plasma creatinine values were lower in the hyperoxia group during resuscitation coinciding with significantly improved renal mitochondrial respiratory capacity and lower 3-nitrotyrosine formation. CONCLUSIONS: Hyperoxia during resuscitation from hemorrhagic shock in swine with preexisting coronary artery disease reduced renal dysfunction and cardiac injury, potentially resulting in improved survival, most likely due to increased mitochondrial respiratory capacity and decreased oxidative and nitrosative stress. Compared with our previous study, the present results suggest a higher benefit of hyperoxia in comorbid swine due to an increased susceptibility to hemorrhagic shock.

Effects of Hyperoxia and Mild Therapeutic Hypothermia During Resuscitation From Porcine Hemorrhagic Shock.

OBJECTIVE: Hemorrhagic shock-induced tissue hypoxia induces hyperinflammation, ultimately causing multiple organ failure. Hyperoxia and hypothermia can attenuate tissue hypoxia due to increased oxygen supply and decreased demand, respectively. Therefore, we tested the hypothesis whether mild therapeutic hypothermia and hyperoxia would attenuate postshock hyperinflammation and thereby organ dysfunction. DESIGN: Prospective, controlled, randomized study. SETTING: University animal research laboratory. SUBJECTS: Thirty-six Bretoncelles-Meishan-Willebrand pigs of either gender. INTERVENTIONS: After 4 hours of hemorrhagic shock (removal of 30% of the blood volume, subsequent titration of mean arterial pressure at 35 mm Hg), anesthetized and instrumented pigs were randomly assigned to "control" (standard resuscitation: retransfusion of shed blood, fluid resuscitation, norepinephrine titrated to maintain mean arterial pressure at preshock values, mechanical ventilation titrated to maintain arterial oxygen saturation > 90%), "hyperoxia" (standard resuscitation, but FIO2, 1.0), "hypothermia" (standard resuscitation, but core temperature 34°C), or "combi" (hyperoxia plus hypothermia) (n = 9 each). MEASUREMENTS AND MAIN RESULTS: Before, immediately at the end of and 12 and 22 hours after hemorrhagic shock, we measured hemodynamics, blood gases, acid-base status, metabolism, organ function, cytokine production, and coagulation. Postmortem kidney specimen were taken for histological evaluation, immunohistochemistry (nitrotyrosine, cystathionine γ-lyase, activated caspase-3, and extravascular albumin), and immunoblotting (nuclear factor-κB, hypoxia-inducible factor-1α, heme oxygenase-1, inducible nitric oxide synthase, B-cell lymphoma-extra large, and protein expression of the endogenous nuclear factor-κB inhibitor). Although hyperoxia alone attenuated the postshock hyperinflammation and thereby tended to improve visceral organ function, hypothermia and combi treatment had no beneficial effect. CONCLUSIONS: During resuscitation from near-lethal hemorrhagic shock, hyperoxia attenuated hyperinflammation, and thereby showed a favorable trend toward improved organ function. The lacking efficacy of hypothermia was most likely due to more pronounced barrier dysfunction with vascular leakage-induced circulatory failure.

Hyperglycemia, oxidative stress, and the diaphragm: a link between chronic co-morbidity and acute stress?

It is well established that prolonged, controlled mechanical ventilation is associated with contractile dysfunction of the diaphragm due to impaired function of the mitochondrial respiratory chain as a result of aggravated oxidative and nitrosative stress. Sepsis and circulatory failure induce a similar response pattern. Callahan and Supinski now show that streptozotocin-induced insulin-dependent diabetes causes a comparable response pattern, both with respect to function and physiology - that is, reduced fiber force and, consequently, muscle contractility - but also as far as the underlying mechanisms are concerned. In other words, the authors elegantly demonstrate that the consequences of a chronic metabolic disease and that of acute critical illness may lead to the same phenotype response. It remains to be elucidated whether the underlying co-morbidity (for example, diabetes) adds to or even synergistically enhances the effect of an acute stress situation (for example, sepsis, mechanical ventilation). In addition, extending their previous work during shock states, the authors also show that administration of a preparation of the enzymatic anti-oxidant superoxide dismutase can reverse the deleterious effects of diabetes. These data are discussed in the context of the fundamental role of hyperglycemia in relation to metabolism-dependent formation of reactive oxygen species.

Effects of pretreatment hypothermia during resuscitated porcine hemorrhagic shock.

OBJECTIVES: Accidental hypothermia increases mortality and morbidity after hemorrhage, but controversial data are available on the effects of therapeutic hypothermia. Therefore, we tested the hypothesis whether moderate pretreatment hypothermia would beneficially influence organ dysfunction during long-term, porcine hemorrhage and resuscitation. DESIGN: Prospective, controlled, randomized study. SETTING: University animal research laboratory. SUBJECTS: Twenty domestic pigs of either gender. INTERVENTIONS: Using an extracorporeal heat exchanger, anesthetized and instrumented animals were maintained at 38°C, 35°C, or 32°C core temperature and underwent 4 hours of hemorrhage (removal of 40% of the blood volume and subsequent blood removal/retransfusion to maintain mean arterial pressure at 30 mm Hg). Resuscitation comprised of hydroxyethyl starch and norepinephrine infusion titrated to maintain mean arterial pressure at preshock values. MEASUREMENTS AND MAIN RESULTS: Before, immediately at the end of, and 12 and 22 hours after hemorrhage, we measured systemic and regional hemodynamics (portal vein, hepatic and right kidney artery ultrasound flow probes) and oxygen transport, and nitric oxide and cytokine production. Hemostasis was assessed by rotation thromboelastometry. Postmortem biopsies were analyzed for histomorphology (hematoxylin and eosin staining) and markers of apoptosis (kidney Bcl-xL and caspase-3 expression). Hypothermia at 32°C attenuated the shock-related lactic acidosis but caused metabolic acidosis, most likely resulting from reduced carbohydrate oxidation. Although hypothermia did not further aggravate shock-related coagulopathy, it caused a transitory attenuation of kidney and liver dysfunction, which was ultimately associated with reduced histological damage and more pronounced apoptosis. CONCLUSIONS: During long-term porcine hemorrhage and resuscitation, moderate pretreatment hypothermia was associated with a transitory attenuation of organ dysfunction and less severe histological tissue damage despite more pronounced metabolic acidosis. This effect is possibly due to a switch from necrotic to apoptotic cell death, ultimately resulting from reduced tissue energy deprivation during the shock phase.

Preserving spontaneous breathing during mechanical ventilatory support: an old yet fascinating story.

Facilitation of early spontaneous breathing activity is the most important measure to shorten weaning and avoid ventilator-induced lung injury and diaphragmatic injury in mechanically ventilated patients. However, the optimal degree of spontaneous muscle activity and ventilator support remains to be determined. Furthermore, effectiveness in relation to the pathophysiology of respiratory failure is unclear. In this regard the experimental study by Saddy and colleagues reveals interesting insights into the pathophysiology of ventilator-induced injury. More important, their results raise important questions that should be evaluated in further studies.

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.

Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock*.

OBJECTIVE: Controversial data are available on the effects of hydrogen sulfide during hemorrhage. Because the clinical significance of hydrogen sulfide administration in rodents may not be applicable to larger species, we tested the hypothesis whether intravenous Na2S (sulfide) would beneficially influence organ dysfunction during long-term, porcine hemorrhage and resuscitation. DESIGN: Prospective, controlled, randomized study. SETTING: University animal research laboratory. SUBJECTS: Forty-five domestic pigs of either gender. INTERVENTIONS: Anesthetized and instrumented animals underwent 4 hrs of hemorrhage (removal of 40% of the blood volume and subsequent blood removal/retransfusion to maintain mean arterial pressure at 30 mm Hg). Sulfide infusion was started 2 hrs before hemorrhage, simultaneously with blood removal or at the beginning of retransfusion of shed blood, and continued for 12 hrs. Resuscitation comprised hydroxyethyl starch and norepinenephrine infusion titrated to maintain mean arterial pressure at preshock values. MEASUREMENTS AND MAIN RESULTS: Before, immediately at the end of and 12 and 22 hrs after hemorrhage, we measured systemic and regional hemodynamics (portal vein, hepatic and right kidney artery ultrasound flow probes) and oxygen transport, nitric oxide and cytokine production (nitrate+nitrite, interleukin-6, tumor necrosis factor-α levels). Postmortem biopsies were analyzed for histomorphology (hematoxylin and eosin staining) and DNA damage (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining). The progressive kidney (creatinine levels, creatinine clearance), liver (transaminase activities, bilirubin levels), and cardiocirculatory (norepipnehrine requirements, troponin I levels) dysfunction was attenuated in the simultaneous treatment group only, which coincided with reduced lung, liver, and kidney histological damage. Sulfide reduced mortality, however, irrespective of the timing of its administration. CONCLUSIONS: While the sulfide-induced protection against organ injury was only present when initiated simultaneously with blood removal, it was largely unrelated to hypothermia. The absence of sulfide-mediated protection in the pretreatment protocol may be due to the accumulation of sulfide during low flow states. In conclusion, sulfide treatment can be effective in hemorrhagic shock, but its effectiveness is restricted to a narrow timing and dosing window.

Endogenous H2S in hemorrhagic shock: innocent bystander or central player?

The role of the gaseous mediator hydrogen sulfide (H2S) in hemorrhagic shock is still a matter of debate. This debate is emphasized by the fact that available literature data on blood and tissue H2S concentrations vary by three orders of magnitude, both under physiological conditions as well as during stress states. Therefore, in a rat model of unresuscitated, lethal hemorrhagic shock, Van de Louw and Haouzi tested the two hypotheses of whether blood and tissue H2S levels would increase due to the shock-related tissue hypoxia, and whether vitamin B12 would attenuate organ injury and improve survival as a result of enhanced H2S oxidation. Hemorrhage did not affect the blood and tissue H2S content, and, despite the increased capacity to oxidize H2S, vitamin B12 did not affect any parameter of shock severity. The authors concluded that H2S concentrations cannot be used as a marker of shock, most probably as a result of tissue's capacity to oxidize H2S even under conditions of severe oxygen debt. This research paper elegantly re-adjusts the currently available data on blood and tissue H2S levels, and thereby adds an important piece to the puzzle of whether H2S release should be enhanced or lowered during stress conditions associated with tissue hypoxia.

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.

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.