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 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.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. 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.

The pentose phosphate pathway (PPP) plays a key role in the cellular regulation of immune function; however, little is known about the interplay of metabolic adjustments in granulocytes, especially regarding the non-oxidative PPP. For the determination of metabolic mechanisms within glucose metabolism, we propose a novel set of measures for ¹³C-metabolic flux analysis based on ex vivo parallel tracer experiments ([1,2-¹³C]glucose, [U-¹³C]glucose, [4,5,6-¹³C]glucose) and gas chromatography–mass spectrometry labeling measurements of intracellular metabolites, such as sugar phosphates and their fragments. A detailed constraint analysis showed that the permission range for net and irreversible fluxes was limited to a three-dimensional space. The overall workflow, including its Bayesian flux estimation, resulted in precise flux distributions and pairwise confidence intervals, some of which could be represented as a line due to the strength of their correlation. The principal component analysis that was enabled by these behaviors comprised three components that explained 99.6% of the data variance. It showed that phagocytic stimulation reversed the direction of non-oxidative PPP net fluxes from ribose-5-phosphate biosynthesis toward glycolytic pathways. This process was closely associated with the up-regulation of the oxidative PPP to promote the oxidative burst.

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 < P_aO₂ < 250 mmHg vs. normoxemia 80 < P_aO₂ < 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.

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.

Traumatic brain injury (TBI) remains one of the leading causes of death. Because of the individual nature of the trauma (brain, circumstances and forces), humans experience individual TBIs. This makes it difficult to generalise therapies. Clinical management issues such as whether intracranial pressure (ICP), cerebral perfusion pressure (CPP) or decompressive craniectomy improve patient outcome remain partly unanswered. Experimental drug approaches for the treatment of secondary brain injury (SBI) have not found clinical application. The complex, cellular and molecular pathways of SBI remain incompletely understood, and there are insufficient experimental (animal) models that reflect the pathophysiology of human TBI to develop translational therapeutic approaches. Therefore, we investigated different injury patterns after acute subdural hematoma (ASDH) as TBI in a post-hoc approach to assess the impact on SBI in a long-term, human-sized porcine TBI animal model. Post-mortem brain tissue analysis, after ASDH, bilateral ICP, CPP, cerebral oxygenation and temperature monitoring, and biomarker analysis were performed. Extracerebral, intraparenchymal–extraventricular and intraventricular blood, combined with brainstem and basal ganglia injury, influenced the experiment and its outcome. Basal ganglia injury affects the duration of the experiment. Recognition of these different injury patterns is important for translational interpretation of results in this animal model of SBI after TBI.

Early life stress (ELS) significantly increases the risk of chronic cardiovascular diseases and may cause neuroinflammation. This post hoc study, based on the material available from a previous study showing elevated “serum brain injury markers” in male control animals, examines the effect of sex and/or ELS on the cerebral and cardiac expression of the H₂S and oxytocin systems. Following approval by the Regional Council of Tübingen, a randomized controlled study was conducted on 12 sexually mature, uncastrated German Large White swine of both sexes. The control animals were separated from their mothers at 28–35 days, while the ELS group was separated at day 21. At 20–24 weeks, animals underwent anesthesia, ventilation, and surgical instrumentation. An immunohistochemical analysis of oxytocin, its receptor, and the H₂S-producing enzymes cystathionine-β-synthase and cystathionine-γ-lyase was performed on hypothalamic, prefrontal cortex, and myocardial tissue samples. Data are expressed as the % of positive tissue staining, and differences between groups were tested using a two-way ANOVA. The results showed no significant differences in the oxytocin and H₂S systems between groups; however, sex influenced the oxytocin system, and ELS affected the oxytocin and H₂S systems in a sex-specific manner. No immunohistochemical correlate to the elevated “serum brain injury markers” in male controls was identified.