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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-β-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.

IntroductionSupplementation 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.MethodsSwine 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).Results13C-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.ConclusionIn 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.

Chronic heart failure is associated with reduced myocardial β-adrenergic receptor expression and mitochondrial function. Since these data coincide with increased plasma catecholamine levels, we investigated the relation between myocardial β-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 (β2-adrenoreceptor, mitochondrial respiration complex, and nitrotyrosine expression). Catecholamine concentrations were inversely related to myocardial mitochondrial respiratory activity and β2-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 β2-adrenoceptor expression and mitochondrial respiration.

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.

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.

IntroductionSodium 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.MethodsAfter 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).ResultsPBMCs showed significantly higher mitochondrial O2 uptake and lower O2•− 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.ConclusionIn 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. O2•− 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.

Trauma and its related psychological and somatic consequences are associated with higher cardiovascular morbidity. The regulation of both the gasotransmitter hydrogen sulfide (H2S) and the neuropeptide oxytocin (OT) have been reported to be affected during physical and psychological trauma. Both mediators are likely molecular correlates of trauma-induced cardiovascular complications, because they share parallel roles and signaling pathways in the cardiovascular system, both locally as well as on the level of central regulation and the vagus nerve. Meditation can alter the structure of specific brain regions and can have beneficial effects on cardiovascular health. This perspective article summarizes the evidence pointing toward the significance of H2S and OT signaling in meditation-mediated cardio-protection.

IntroductionWe 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.MethodsAfter 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.ResultsNor 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.ConclusionWhether 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.

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.

BackgroundThe hydrogen sulfide (H2S) and the oxytocin/oxytocin receptor (OT/OTR) systems interact in the central nervous and cardiovascular system. As a consequence of osmotic balance stress, H2S stimulates OT release from the paraventricular nuclei (PVN) in the hypothalamic regulation of blood volume and pressure. Hemorrhagic shock (HS) represents one of the most pronounced acute changes in blood volume, which, moreover, may cause at least transient brain tissue hypoxia. Atherosclerosis is associated with reduced vascular expression of the main endogenous H2S producing enzyme cystathionine-γ-lyase (CSE), and, hence, exogenous H2S administration could be beneficial in these patients, in particular after HS. However, so far cerebral effects of systemic H2S administration are poorly understood. Having previously shown lung-protective effects of therapeutic Na2S2O3 administration in a clinically relevant, long-term, porcine model of HS and resuscitation we evaluated if these protective effects were extended to the brain.MethodsIn this study, available unanalyzed paraffin embedded brain sections (Na2S2O3N = 8 or vehicle N = 5) of our recently published HS study were analyzed via neuro-histopathology and immunohistochemistry for the endogenous H2S producing enzymes, OT, OTR, and markers for brain injury and oxidative stress (glial fibrillary acidic protein (GFAP) and nitrotyrosine).ResultsNeuro-histopathological analysis revealed uninjured brain tissue with minor white matter edema. Protein quantification in the hypothalamic PVN showed no significant inter-group differences between vehicle or Na2S2O3 treatment.ConclusionsThe endogenous H2S enzymes, OT/OTR co-localized in magnocellular neurons in the hypothalamus, which may reflect their interaction in response to HS-induced hypovolemia. The preserved blood brain barrier (BBB) may have resulted in impermeability for Na2S2O3 and no inter-group differences in the PVN. Nonetheless, our results do not preclude that Na2S2O3 could have a therapeutic benefit in the brain in an injury that disrupts the BBB, e.g., traumatic brain injury (TBI) or acute subdural hematoma (ASDH).

In the porcine model discussed in this review, the acute subdural hematoma was induced by subdural injection of autologous blood over the left parietal cortex, which led to a transient elevation of the intracerebral pressure, measured by bilateral neuromonitoring. The hematoma-induced brain injury was associated with albumin extravasation, oxidative stress, reactive astrogliosis and microglial activation in the ipsilateral hemisphere. Further proteins and injury markers were validated to be used for immunohistochemistry of porcine brain tissue. The cerebral expression patterns of oxytocin, oxytocin receptor, cystathionine-γ-lyase and cystathionine-β-synthase were particularly interesting: these four proteins all co-localized at the base of the sulci, where pressure-induced brain injury elicits maximum stress. In this context, the pig is a very relevant translational model in contrast to the rodent brain. The structure of the porcine brain is very similar to the human: the presence of gyri and sulci (gyrencephalic brain), white matter to grey matter proportion and tentorium cerebelli. Thus, pressure-induced injury in the porcine brain, unlike in the rodent brain, is reflective of the human pathophysiology.

Abstract Glucocorticoids (GCs) are stress hormones that regulate developmental and physiological processes and are among the most potent anti-inflammatory drugs to suppress chronic and acute inflammation. GCs act through the glucocorticoid receptor (GR), a ubiquitously expressed ligand-activated transcription factor, which translocates into the nucleus and can act via two different modes, as a GR monomer or as a GR dimer. These two modes of action are not clearly differentiated in practice and may lead to completely different therapeutic outcomes. Detailed aspects of GR mechanisms are often not taken into account when GCs are used in different clinical scenarios. Patients, with critical illness-related corticosteroid insufficiency, treated with natural or synthetic GCs are still missing a clearly defined therapeutic strategy. This review discusses the different modes of GR function and its importance on organ function in vivo.

Abstract This review addresses the plausibility of hydrogen sulfide (H2S) therapy for acute lung injury (ALI) and circulatory shock, by contrasting the promising preclinical results to the present clinical reality. The review discusses how the narrow therapeutic window and width, and potentially toxic effects, the route, dosing, and timing of administration all have to be balanced out very carefully. The development of standardized methods to determine in vitro and in vivo H2S concentrations, and the pharmacokinetics and pharmacodynamics of H2S-releasing compounds is a necessity to facilitate the safety of H2S-based therapies. We suggest the potential of exploiting already clinically approved compounds, which are known or unknown H2S donors, as a surrogate strategy.

BackgroundSodium 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.MethodsUnder 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.ResultsDiabetes 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.ConclusionIn 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.

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.

Circulatory shock is associated with marked disturbances of the macro- and microcirculation and flow heterogeneities. Furthermore, a lack of tissue adenosine trisphosphate (ATP) and mitochondrial dysfunction are directly associated with organ failure and poor patient outcome. While it remains unclear if microcirculation-targeted resuscitation strategies can even abolish shock-induced flow heterogeneity, mitochondrial dysfunction and subsequently diminished ATP production could still lead to organ dysfunction and failure even if microcirculatory function is restored or maintained. Preserved mitochondrial function is clearly associated with better patient outcome. This review elucidates the role of the microcirculation and mitochondria during circulatory shock and patient management and will give a viewpoint on the advantages and disadvantages of tailoring resuscitation to microvascular or mitochondrial targets.

The hydrogen sulfide (H2S) and the oxytocin/oxytocin receptor (OT/OTR) systems interact in trauma and are implicated in vascular protection and regulation of fluid homeostasis. Acute brain injury is associated with pressure-induced edema formation, blood brain barrier disruption, and neuro-inflammation. The similarities in brain anatomy: size, gyrencephalic organization, skull structure, may render the pig a highly relevant model for translational medicine. Cerebral biomarkers for pigs for pathophysiological changes and neuro-inflammation are limited. The current study aims to characterize the localization of OT/OTR and the endogenous H2S producing enzymes together with relevant neuro-inflammatory markers on available porcine brain tissue from an acute subdural hematoma (ASDH) model. In a recent pilot study, anesthetized pigs underwent ASDH by injection of 20 mL of autologous blood above the left parietal cortex and were resuscitated with neuro-intensive care measures. After 54 h of intensive care, the animals were sacrificed, the brain was removed and analyzed via immunohistochemistry. The endogenous H2S producing enzymes cystathionine-ɤ-lyase (CSE) and cystathionine-β-synthase (CBS), the OTR, and OT were localized in neurons, vasculature and parenchyma at the base of sulci, where pressure-induced injury leads to maximal stress in the gyrencephalic brain. The pathophysiological changes in response to brain injury in humans and pigs, we show here, are comparable. We additionally identified modulators of brain injury to further characterize the pathophysiology of ASDH and which may indicate future therapeutic approaches.

The pathophysiology of sepsis-induced myocardial dysfunction is not resolved to date and comprises inflammation, barrier dysfunction and oxidative stress. Disease-associated reduction of tissue cystathionine-γ-lyase (CSE) expression, an endogenous H2S-producing enzyme, is associated with oxidative stress, barrier dysfunction and organ injury. CSE-mediated cardio-protection has been suggested to be related the upregulation of oxytocin receptor (OTR). CSE can also mediate glucocorticoid receptor (GR) signaling, which is important for normal heart function. A sepsis-related loss of cardiac CSE expression associated with impaired organ function has been reported previously. The aim of this current post hoc study was to investigate the role of cardiac GR and OTR after polymicrobial sepsis in a clinically relevant, resuscitated, atherosclerotic porcine model. Anesthetized and instrumented FBM (Familial Hypercholesterolemia Bretoncelles Meishan) pigs with high fat diet-induced atherosclerosis underwent poly-microbial septic shock (n = 8) or sham procedure (n = 5), and subsequently received intensive care therapy with fluid and noradrenaline administration for 24 h. Cardiac protein expression and mRNA levels were analyzed. Systemic troponin, a marker of cardiac injury, was significantly increased in septic animals in contrast to sham, whereas OTR and GR expression in septic hearts were reduced, along with a down-regulation of anti-inflammatory GR target genes and the antioxidant transcription factor NRF2. These results suggest a potential interplay between GR, CSE, and OTR in sepsis-mediated oxidative stress, inflammation and cardiac dysfunction.

Background: Sepsis, that can be modeled by LPS injections, as an acute systemic inflammation syndrome is the most common cause for acute lung injury (ALI). ALI induces acute respiratory failure leading to hypoxemia, which is often associated with multiple organ failure (MOF). During systemic inflammation, the hypothalamus-pituitary-adrenal axis (HPA) is activated and anti-inflammatory acting glucocorticoids (GCs) are released to overcome the inflammation. GCs activate the GC receptor (GR), which mediates its effects via a GR monomer or GR dimer. The detailed molecular mechanism of the GR in different inflammatory models and target genes that might be crucial for resolving inflammation is not completely identified. We previously observed that mice with attenuated GR dimerization (GRdim/dim) had a higher mortality in a non-resuscitated lipopolysaccharide (LPS)- and cecal ligation and puncture (CLP)-induced inflammation model and are refractory to exogenous GCs to ameliorate ALI during inflammation. Therefore, we hypothesized that impaired murine GR dimerization (GRdim/dim) would further impair organ function in LPS-induced systemic inflammation under human like intensive care management and investigated genes that are crucial for lung function in this setup.Methods: Anesthetized GRdim/dim and wildtype (GR+/+) mice were challenged with LPS (10 mg·kg−1, intraperitoneal) and underwent intensive care management (“lung-protective” mechanical ventilation, crystalloids, and norepinephrine) for 6 h. Lung mechanics and gas exchange were assessed together with systemic hemodynamics, acid-base status, and mitochondrial oxygen consumption (JO2). Western blots, immunohistochemistry, and real time quantitative polymerase chain reaction were performed to analyze lung tissue and inflammatory mediators were analyzed in plasma and lung tissue.Results: When animals were challenged with LPS and subsequently resuscitated under intensive care treatment, GRdim/dim mice had a higher mortality compared to GR+/+ mice, induced by an increased need of norepinephrine to achieve hemodynamic targets. After challenge with LPS, GRdim/dim mice also displayed an aggravated ALI shown by a more pronounced impairment of gas exchange, lung mechanics and increased osteopontin (Opn) expression in lung tissue.Conclusion: Impairment of GR dimerization aggravates systemic hypotension and impairs lung function during LPS-induced endotoxic shock in mice. We demonstrate that the GR dimer is an important mediator of hemodynamic stability and lung function, possibly through regulation of Opn, during LPS-induced systemic inflammation.

Abstract Background Sepsis is associated with disturbed glucose metabolism and reduced mitochondrial activity and biogenesis, ultimately leading to multiple organ dysfunction, e.g., acute kidney injury (AKI). Cystathionine-γ-lyase (CSE), the major cardiovascular source of endogenous H2S release, is implicated in the regulation of glucose metabolism and mitochondrial activity through a PGC1α-dependent mechanism, and critical for kidney function. Atherosclerosis is associated with mitochondrial dysfunction and reduced CSE expression. Thus, the aim of this post hoc study was to test the hypothesis whether there is an interplay between CSE expression and kidney dysfunction, mitochondrial activity, and oxidative/nitrosative stress in porcine septic AKI with underlying coronary artery disease. Methods This study is a post hoc analysis of material from anesthetized and instrumented swine with a high fat diet-induced hypercholesterolemia and atherosclerosis undergoing faecal peritonitis-induced septic shock or sham procedure and intensive care (comprising fluid resuscitation and continuous i.v. noradrenaline (NoA) infusion) for 24 h. Glucose metabolism was quantified from blood 13C6-glucose and expiratory 13CO2/12CO2 isotope enrichment during 13C6-glucose infusion. Mitochondrial activity was determined by high-resolution respirometry. CSE and PGC1α expression, as well as nitrotyrosine formation and albumin extravasation, were quantified by immunohistochemistry of formalin-fixed kidney paraffin sections. Results Sepsis was associated with lactic acidosis (p = 0.004) and AKI (50% fall of creatinine clearance (CrCl), p = 0.019). While both whole-body glucose production (p = 0.004) and oxidation (p = 0.006) were increased, kidney tissue mitochondrial respiration was reduced (p = 0.028), coinciding with decreased CSE (p = 0.003) and PGC1α (p = 0.003) expression. Albumin extravasation (p = 0.011) and nitrotyrosine formation (p = 0.008) were increased in septic kidneys. Conclusions Sepsis-induced AKI is associated with disturbed mitochondrial respiration and biogenesis, which may be aggravated by oxidative and nitrosative stress. Our results confirm previous data in murine septic shock and porcine hemorrhage and resuscitation on the crucial role of CSE for barrier integrity and kidney function.

Abstract Background Both the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) and oxytocin/oxytocin receptor (OT/OTR) systems have been reported to be cardioprotective. H2S can stimulate OT release, thereby affecting blood volume and pressure regulation. Systemic hyper-inflammation after blunt chest trauma is enhanced in cigarette smoke (CS)-exposed CSE−/− mice compared to wildtype (WT). CS increases myometrial OTR expression, but to this point, no data are available on the effects CS exposure on the cardiac OT/OTR system. Since a contusion of the thorax (Txt) can cause myocardial injury, the aim of this post hoc study was to investigate the effects of CSE−/− and exogenous administration of GYY4137 (a slow release H2S releasing compound) on OTR expression in the heart, after acute on chronic disease, of CS exposed mice undergoing Txt. Methods This study is a post hoc analysis of material obtained in wild type (WT) homozygous CSE−/− mice after 2-3 weeks of CS exposure and subsequent anesthesia, blast wave-induced TxT, and surgical instrumentation for mechanical ventilation (MV) and hemodynamic monitoring. CSE−/− animals received a 50 μg/g GYY4137-bolus after TxT. After 4h of MV, animals were exsanguinated and organs were harvested. The heart was cut transversally, formalin-fixed, and paraffin-embedded. Immunohistochemistry for OTR, arginine-vasopressin-receptor (AVPR), and vascular endothelial growth factor (VEGF) was performed with naïve animals as native controls. Results CSE−/− was associated with hypertension and lower blood glucose levels, partially and significantly restored by GYY4137 treatment, respectively. Myocardial OTR expression was reduced upon injury, and this was aggravated in CSE−/−. Exogenous H2S administration restored myocardial protein expression to WT levels. Conclusions This study suggests that cardiac CSE regulates cardiac OTR expression, and this effect might play a role in the regulation of cardiovascular function.

This paper explored the potential mediating role of hydrogen sulfide (H2S) and the oxytocin (OT) systems in hemorrhagic shock (HS) and/or traumatic brain injury (TBI). Morbidity and mortality after trauma mainly depend on the presence of HS and/or TBI. Rapid “repayment of the O2 debt” and prevention of brain tissue hypoxia are cornerstones of the management of both HS and TBI. Restoring tissue perfusion, however, generates an ischemia/reperfusion (I/R) injury due to the formation of reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, pre-existing-medical-conditions (PEMC’s) can aggravate the occurrence and severity of complications after trauma. In addition to the “classic” chronic diseases (of cardiovascular or metabolic origin), there is growing awareness of psychological PEMC’s, e.g., early life stress (ELS) increases the predisposition to develop post-traumatic-stress-disorder (PTSD) and trauma patients with TBI show a significantly higher incidence of PTSD than patients without TBI. In fact, ELS is known to contribute to the developmental origins of cardiovascular disease. The neurotransmitter H2S is not only essential for the neuroendocrine stress response, but is also a promising therapeutic target in the prevention of chronic diseases induced by ELS. The neuroendocrine hormone OT has fundamental importance for brain development and social behavior, and, thus, is implicated in resilience or vulnerability to traumatic events. OT and H2S have been shown to interact in physical and psychological trauma and could, thus, be therapeutic targets to mitigate the acute post-traumatic effects of chronic PEMC’s. OT and H2S both share anti-inflammatory, anti-oxidant, and vasoactive properties; through the reperfusion injury salvage kinase (RISK) pathway, where their signaling mechanisms converge, they act via the regulation of nitric oxide (NO).

Abstract Background Cystathionine-γ-lyase (CSE) was shown to have a regulatory role in glucose metabolism. Circulatory shock can induce metabolic stress, thereby leading to hyperglycemia and mitochondrial dysfunction. In vitro data suggest an effect of high glucose on CSE expression. Therefore, the aim of this study was to investigate the effects of hyperglycemia on CSE expression in resuscitated murine septic shock. Methods Normo- (80–150 mg/dl) and hyperglycemic (>200 mg/dl) male C57/BL6J mice (n = 5-6 per group) underwent cecal ligation and puncture (CLP)-induced polymicrobial sepsis or sham procedure (n = 6 per group) and, 15 h afterwards, were anesthetized again, surgically instrumented and received intensive care treatment, including antibiotics, lung protective mechanical ventilation, circulatory support, and intravenous (i.v.) glucose infusion (50% as stable-isotope labeled 1,2,3,4,5,6–13C6 glucose). Blood and breath gas were sampled hourly to quantify parameters of glucose metabolism. 5 h later, mice were sacrificed and organs were harvested. The liver mitochondrial respiratory activity was determined via high resolution respirometry; CSE, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), and adipocyte differentiation-related protein (ADRP) expression was immunohistochemically investigated. Results In sepsis combined with hyperglycemia the least CSE and PGC1α expression could be detected, along with reduced mitochondrial respiratory activity, and enhanced ADRP expression, a marker of lipid droplet formation, in the liver. A novel in vivo finding is the CSE translocation from the cytosol to the nucleus triggered by metabolic stress. Conclusions A relationship between CSE and glucose metabolism was established, which, when dysregulated, may contribute to fatty liver disease and hepatic steatosis.

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.

The purpose of this review is to explore the parallel roles and interaction of hydrogen sulfide (H2S) and oxytocin (OT) in cardiovascular regulation and fluid homeostasis. Their interaction has been recently reported to be relevant during physical and psychological trauma. However, literature reports on H2S in physical trauma and OT in psychological trauma are abundant, whereas available information regarding H2S in psychological trauma and OT in physical trauma is much more limited. This review summarizes recent direct and indirect evidence of the interaction of the two systems and their convergence in downstream nitric oxide-dependent signaling pathways during various types of trauma, in an effort to better understand biological correlates of psychosomatic interdependencies.

Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.