Effect of mitoTEMPO on Redox Reactions in Different Body Compartments upon Endotoxemia in Rats.

Mitochondrial ROS (mitoROS) control many reactions in cells. Biological effects of mitoROS in vivo can be investigated by modulation via mitochondria-targeted antioxidants (mtAOX, mitoTEMPO). The aim of this study was to determine how mitoROS influence redox reactions in different body compartments in a rat model of endotoxemia. We induced inflammatory response by lipopolysaccharide (LPS) injection and analyzed effects of mitoTEMPO in blood, abdominal cavity, bronchoalveolar space, and liver tissue. MitoTEMPO decreased the liver damage marker aspartate aminotransferase; however, it neither influenced the release of cytokines (e.g., tumor necrosis factor, IL-4) nor decreased ROS generation by immune cells in the compartments examined. In contrast, ex vivo mitoTEMPO treatment substantially reduced ROS generation. Examination of liver tissue revealed several redox paramagnetic centers sensitive to in vivo LPS and mitoTEMPO treatment and high levels of nitric oxide (NO) in response to LPS. NO levels in blood were lower than in liver, and were decreased by in vivo mitoTEMPO treatment. Our data suggest that (i) inflammatory mediators are not likely to directly contribute to ROS-mediated liver damage and (ii) mitoTEMPO is more likely to affect the redox status of liver cells reflected in a redox change of paramagnetic molecules. Further studies are necessary to understand these mechanisms.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

EPR analysis of extra- and intracellular nitric oxide in liver biopsies.

PURPOSE: To develop an assay that can enable the quantification of intra- and extracellular nitric oxide (NO) levels in liver biopsies without application of potentially harmful exogenous NO traps. THEORY: Electron paramagnetic resonance (EPR) spectroscopy is currently the most appropriate method of measuring NO in biological samples due to the outstanding specificity resulting from the interaction of NO with exogenous NO traps. Because such traps are not allowed in clinical settings, we tested the reliability of endogenous NO traps for the determination of NO levels in blood and liver compartments. METHODS: Rats were injected with 0-8 mg/kg lipopolysaccharide (LPS) to gradually induce a systemic inflammatory response. Specific features of NO-hemoglobin and NO-Fe EPR signals were quantified using a specifically developed calibration procedure. RESULTS: Whereas both NO-hemoglobin (NO-HbLIVER BLOOD ) and NO-Fe (NO-FeLIVER ) complexes were detected in nonperfused liver tissue, only NO-Fe complexes were detected in perfused tissue and only NO-Hb complexes were detected in blood (NO-HbBLOOD ). The NO concentrations increased in the sequence NO-HbBLOOD < NO-FeLIVER < NO-HbLIVER BLOOD (9.4, 18.5, 27.9 nmol/cm3 , respectively at 2.5 mg/kg LPS). The detection limit of the method was 0.61 nmol/cm3 for NO-Hb and 0.52 nmol/cm3 for NO-Fe. CONCLUSION: The assay reported here does not influence natural NO pathways and enables the quantification of NO distribution in two liver compartments using a single liver biopsy. Magn Reson Med 77:2372-2380, 2017. © 2016 International Society for Magnetic Resonance in Medicine.