Min Of Reoxygenation Research Articles

Mitochondrial responses to constant and cyclic hypoxia depend on the oxidized fuel in a hypoxia-tolerant marine bivalve Crassostrea gigas

Sessile benthic organisms like oysters inhabit the intertidal zone, subject to alternating hypoxia and reoxygenation (H/R) episodes during tidal movements, impacting respiratory chain activities and metabolome compositions. We investigated the effects of constant severe hypoxia (90 min at ~ 0% O2 ) followed by 10 min reoxygenation, and cyclic hypoxia (5 cycles of 15 min at ~ 0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of Crassostrea gigas respiring on pyruvate, palmitate, or succinate. Constant hypoxia suppressed oxidative phosphorylation (OXPHOS), particularly during Complex I-linked substrates oxidation. It had no effect on mitochondrial reactive oxygen species (ROS) efflux but increased fractional electron leak (FEL). In mitochondria oxidizing Complex I substrates, exposure to cyclic hypoxia prompted a significant drop after the first H/R cycle. In contrast, succinate-driven respiration only showed significant decline after the third to fifth H/R cycle. ROS efflux saw little change during cyclic hypoxia regardless of the oxidized substrate, but Complex I-driven FEL tended to increase with each subsequent H/R cycle. These observations suggest that succinate may serve as a beneficial stress fuel under H/R conditions, aiding in the post-hypoxic recovery of oysters by reducing oxidative stress and facilitating rapid ATP re-synthesis. The impacts of constant and cyclic hypoxia of similar duration on mitochondrial respiration and oxidative lesions in the proteins were comparable indicating that the mitochondrial damage is mostly determined by the lack of oxygen and mitochondrial depolarization. The ROS efflux in the mitochondria of oysters was minimally affected by oxygen fluctuations indicating that tight regulation of ROS production may contribute to robust mitochondrial phenotype of oysters and protect against H/R induced stress.

The effects of CO2 levels and body temperature on brain interstitial pH alterations during the induction of hypoxic-ischemic encephalopathy in newborn pigs

Brain interstitial pH (pHbrain) alterations play a crucial role in the development of hypoxic-ischemic (HI) encephalopathy (HIE) caused by asphyxia in neonates. The newborn pig is one of the most suitable large animal models for studying HIE, however, compared to rats, experimental data on pHbrain alterations during HIE induction are limited. The major objective of the present study was thus to compare pHbrain changes during HIE development induced by experimental normocapnic hypoxia (H) or asphyxia (A), elicited with ventilation of a gas mixture containing 6%O2 or 6%O2/20%CO2, respectively for 20 min, under either normothermia (NT) or hypothermia (HT) (38.5 ± 0.5 °C or 33.5 ± 0.5 °C core temperature, respectively) in anesthetized piglets yielding four groups: H-NT, A-NT, H-HT, and A-HT. pHbrain changes during HI stress and the 60 min reoxygenation period were measured using a pH-selective microelectrode inserted into the parietal cortex through an open cranial window. In all groups, the pHbrain response to HI stress was acidosis, at the nadir pHbrain values dropped from the baseline of 7.27 ± 0.02 to H-NT:5.93 ± 0.30, A-NT:5.90 ± 0.52, H-HT:6.81 ± 0.27, and A-HT:6.27 ± 0.24 indicating that (1) H and A elicited similar, severe brain acidosis under NT greatly exceeding pH changes in arterial blood (pHa dropped to 7.24 ± 0.07 and 6.78 ± 0.03 from 7.52 ± 0.06 and 7.50 ± 0.05, respectively), and (2) HT ameliorated more the brain acidosis induced by H than by A. In all four groups, pHbrain was restored to baseline values without an alkalotic overshoot during the observed reoxygenation, Our findings suggest that under NT either H or A – both commonly employed HI stresses to elicit HIE in piglet models – would result in a similar acidotic pHbrain response without an alkalotic component either during the HI stress or the early reoxygenation period.

Non-neuronal acetylcholine is causal for cardioprotection by hypoxic preconditioning in adult rat cardiomyocytes

Abstract Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia and reperfusion reduce lethal ischemia/reperfusion injury. The underlying protective signaling mechanisms of such ischemic preconditioning are unclear in their details. Among other protective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is involved in protection by ischemic preconditioning. Recently, a non-neuronal cardiac cholinergic system (NNCCS) has been identified, suggesting that a nervous system-independent, cardiomyocyte-derived acetylcholine signaling with signal amplification by auto- and paracrine cholinergic receptor activation could also participate in ischemic preconditioning. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of ischemic preconditioning. We therefore, in a reductionist approach, used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Adult ventricular rat cardiomyocytes were isolated (from n=9 hearts), and HPC was induced by one cycle of 10 min hypoxia / 20 min reoxygenation. Thirty min normoxia served as control. Cardiomyocytes were then exposed to 30 min sustained hypoxia / 5 min reoxygenation (H/R), or a 35 min time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, by trypan blue staining. In a subgroup of experiments (cardiomyocytes from n=6 hearts) in the presence of physostigmine (0.2 mmol/L), intracellular ACh was quantified before and after HPC or normoxia and after sustained hypoxia and reoxygenation or TC, respectively, via liquid chromatography-coupled tandem mass spectrometry. Experiments on cardiomyocyte viability (from n=9 hearts) were repeated with the addition of cholinergic receptor antagonists (nicotinic: hexamethonium [1 µmol/L], muscarinic: atropine [0.1 µmol/L]) a) during the first 30 min with HPC or normoxia, b) during H/R or TC (30-65 min), or c) throughout the total 65 min protocol. Results The viability of cardiomyocytes subjected to H/R was better preserved with HPC than without (Figure A), and intracellular ACh was increased during hypoxia after prior HPC (Figure B). Cholinergic receptor antagonists during HPC did not change HPC´s protection (Figure A). However, the combination of both antagonists during sustained hypoxia abrogated HPC´s protection (Figure A), whereas nicotinic or muscarinic cholinergic receptor antagonists alone did not. Cholinergic receptor antagonists throughout the total 65 min also abrogated HPC´s protection (Figure A). Conclusion: The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular acetylcholine, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an auto- and paracrine signal amplification on the cardiomyocyte level.Figure

Mitochondrial respiration analysis in permeabilized porcine left ventricular and human right atrial specimens with ischemia-reperfusion.

Mitochondrial function is critical to myocardial ischemia-reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison with those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins [cytochrome-c oxidase 4 (COX4), citrate synthase, and manganese-dependent superoxide dismutase]. When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score, -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson's R of 0.87). Mitochondrial dysfunction by ischemia-reperfusion was equally reflected in PMT and isolated mitochondria (44 and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia-reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia-reperfusion.NEW & NOTEWORTHY Methods to quantify mitochondrial function in translationally relevant models and in human tissue are needed to improve the translation of cardioprotection to patients. Our present approach, using PMT instead of isolated mitochondria for the quantification of mitochondrial ischemia-reperfusion damage, provides a reference for further studies in translationally relevant large animal models and in human tissue, thus possibly improving the translation of cardioprotection to the benefit of patients with acute myocardial infarction.

Cholinergic action of spleen‐derived cardioprotective factors

IntroductionThere is an ambivalent relationship between the spleen and the heart in myocardial infarction (Circ Res 124; 2019; 26‐8). Sympathetic activation of the spleen initially increases myocardial inflammatory infiltration but resolves it later by initiating a myocardial healing response. Vagal activation of the spleen induces the release of cardioprotective factors: Perfusion of an isolated rat spleen with the vagomimetic carbachol resulted in the release of factors which then reduced infarct size (IS) in an isolated perfused rat heart.AimTo characterize cardioprotection of spleen‐derived factors, with a focus on their cholinergic action.MethodsRat spleens and hearts were isolated and buffer‐perfused at constant pressure. Isolated spleens were either challenged by infusion of saline (as control) or the vagomimetic carbachol (500 pmol/L), and splenic effluate (SEff; 100 mL) was sampled after saline infusion (SEffsaline) or carbachol infusion (SEffcarbachol), respectively. Cell‐free SEff was prepared by centrifugation and filtration. SEffsaline or SEffcarbachol was infused into isolated hearts which were then subjected to 30 min global ischemia and 120 min reperfusion (I/R). In subsets of experiments, the hearts were pretreated with the nicotinic receptor antagonist hexamethonium (50 µmol/L) or the muscarinic receptor antagonist atropine (100 pmol/L). IS was demarcated by triphenyltetrazolium chloride staining and calculated as percent of ventricular mass. To study the relevance of spleen‐derived protection on the cardiomyocyte level, adult rat ventricular cardiomyocytes were prepared. Cells were incubated for 30 minutes with SEffsaline or SEffcarbachol, respectively, and then subjected to 30 min hypoxia/ 5 min reoxygenation (H/R) or exposed to SEffsaline or SEffcarbachol for 35 minutes as time control (TC). Since nicotinic and muscarinic receptors are also expressed on cardiomyocytes, all experiments were repeated after pretreatment with hexamethonium (1 µmol/L) or atropine (100 nmol/L). Cardiomyocyte viability was quantified before (baseline) and after H/R or TC, as the percent fraction of rod‐shaped, unstained (5% trypan blue) cells over all cells. The estimated final concentration of carbachol in the SEffcarbachol of ≤10 pmol/L and the SEffsaline did neither reduce IS in isolated perfused rat hearts nor improve cardiomyocyte viability in isolated rat cardiomyocytes.ResultsWith SEffsaline, IS was 34±7%. With SEffcarbachol, IS was reduced to 19±8%. Hexamethonium or atropine had no impact on IS per se in hearts with SEffsaline (33±8%; 34±6%), but attenuated the cardioprotection by SEffcarbachol (27±10%; 26±10%; Figure A). The viability of cardiomyocytes subjected to H/R was better preserved with SEffcarbachol than with SEffsaline or saline alone. Again, this protection was attenuated by hexamethonium or atropine (Figure B, insert).ConclusionThe cardioprotection by spleen‐derived factors, which were released in response to the vagomimetic carbachol, is mediated through nicotinic and muscarinic receptors within the heart. Apparently, this protection does not need to involve neuronal structures within the myocardium – nicotinic and muscarinic receptors on cardiomyocytes per se mediate protection.

Neuroprotection in the Striatum of Hypoxic-Ischemic Piglets by Simultaneous Inhibition of Dopamine D1 and Adenosine A2A Receptors

Introduction: Striatal neurons of term newborns are highly vulnerable to hypoxia-ischemia (H-I). In a piglet model of H-I, a dopamine D1 receptor antagonist and an adenosine A2A receptor antagonist alone preferentially protect striatonigral and striatopallidal neurons, respectively. Here, we tested the hypothesis whether the combined treatment with SCH23390, a D1 receptor antagonist, and SCH58261, an A2A receptor antagonist, is more efficacious than individual D1 and A2A receptor antagonist treatment. Methods: Anesthetized newborn piglets were subjected to sham operation (n = 6) or 40 min of hypoxia and 7 min of airway occlusion. At 5 min of reoxygenation, piglets received the vehicle, SCH23390, SCH58261, or the combined treatment (n = 9 in each group). At 4 days of recovery, the number of viable neurons in the entire putamen was estimated by unbiased stereology. Results: Stereological results showed that sham-operated piglets had an estimated 2.9 × 106 neurons in the putamen, and the number of viable neurons in hypoxic-ischemic piglets was significantly reduced by 80% to 0.6 × 106/putamen. Treatment with SCH23390, SCH58261, and the combination increased the numbers of viable neurons to 1.4 × 106/putamen, 1.4 × 106/putamen, and 2.1 × 106/putamen, respectively. Notably, the combined treatment improved neuroprotection compared to individual therapy. Conclusion: We conclude that simultaneous inhibition of dopamine D1 receptors and adenosine A2A receptors saves more neurons than individual treatment in the highly vulnerable putamen of a large-animal neonatal H-I model.

Tissue- and substrate-dependent mitochondrial responses to acute hypoxia–reoxygenation stress in a marine bivalve (Crassostrea gigas )

Hypoxia is a major stressor for aquatic organisms, yet intertidal organisms such as the oyster Crassostrea gigas are adapted to frequent oxygen fluctuations by metabolically adjusting to shifts in oxygen and substrate availability during hypoxia-reoxygenation (H/R). We investigated the effects of acute H/R stress (15 min at ∼0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of C. gigas respiring on different substrates (pyruvate, glutamate, succinate, palmitate and their mixtures). Gill mitochondria showed better capacity for amino acid and fatty acid oxidation compared with mitochondria from the digestive gland. Mitochondrial responses to H/R stress strongly depended on the substrate and the activity state of mitochondria. In mitochondria oxidizing NADH-linked substrates, exposure to H/R stress suppressed oxygen consumption and generation of reactive oxygen species (ROS) in the resting state, whereas in the ADP-stimulated state, ROS production increased despite little change in respiration. As a result, electron leak (measured as H2O2 to O2 ratio) increased after H/R stress in the ADP-stimulated mitochondria with NADH-linked substrates. In contrast, H/R exposure stimulated succinate-driven respiration without an increase in electron leak. Reverse electron transport (RET) did not significantly contribute to succinate-driven ROS production in oyster mitochondria except for a slight increase in the OXPHOS state during post-hypoxic recovery. A decrease in NADH-driven respiration and ROS production, enhanced capacity for succinate oxidation and resistance to RET might assist in post-hypoxic recovery of oysters mitigating oxidative stress and supporting rapid ATP re-synthesis during oxygen fluctuations, as is commonly observed in estuaries and intertidal zones.

Abstract 11004: Activated Protein C Protects Human Cardiomyocytes Against Hypoxia Reoxygenation Stress-Induced Injury Through Maintaining Mitochondrial Integrity

Introduction: Activated protein C (APC) is a vitamin-K-dependent plasma serine protease that functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. Endothelial protein C receptor (EPCR) mediates APC's cytoprotective properties. We found that cardiac EPCR expression is downregulated in aging, leading to cardiac intolerance to ischemic insults. However, the role of APC in the human heart under ischemic stress remains unclear. Hypothesis: APC-EPCR axis protects human cardiomyocytes against hypoxia-reoxygenation stress-induced damage through maintaining mitochondrial integrity. Methods: The left ventricles of 38 adult human donor hearts underwent proteomic analysis of myocardial EPCR. Viable human cardiomyocytes were isolated and challenged by 30 min hypoxia/30 min reoxygenation (H/R) with or without APC treatment. Cardiomyocyte contractile properties were determined by IonOptix Imaging System, while the mitochondrial functional integrity and reactive oxygen species (ROS) were determined with Seahorse XF analyzer and MitoSox staining. Results: Immunoblotting data demonstrated that EPCR expression levels are declined in human hearts along with aging. H/R-stress reduced contractile functions of human cardiomyocytes, and the cardiomyocytes from aged adults (>65 years) versus cardiomyocytes from young adults (<65 years) were more vulnerable to H/R-induced insults. Interestingly, the administration of APC significantly improved the contractile functions of both young and aged human cardiomyocytes under H/R stress. APC also stabilizes EPCR levels and modulates the calcium flux homeostasis in both young and aged human cardiomyocytes under H/R. The mitochondrial respiration experimental data demonstrated that APC treatment ameliorates the mitochondrial dysfunction induced by H/R stress in both young and aged human cardiomyocytes. Since the ROS generations in human cardiomyocytes during H/R stress were attenuated with APC administration. Conclusions: Cardiac EPCR levels are downregulated with aging. The administration of APC can rescue the cardiac intolerance to ischemic insults in aging through stabilizing receptor EPCR and maintaining cardiomyocyte mitochondrial integrity.

Intrinsic Mechanisms Underlying Hypoxia-Tolerant Mitochondrial Phenotype During Hypoxia-Reoxygenation Stress in a Marine Facultative Anaerobe, the Blue Mussel Mytilus edulis

Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue musselMytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating thatM. eduliswas able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.

The Non‐Neuronal Cholinergic System is Causal for Cardioprotection by Hypoxic Preconditioning in Isolated Adult Rat Cardiomyocytes

Introduction Brief episodes of non-lethal myocardial ischemia/reperfusion preceding more sustained ischemia reduce lethal ischemia/reperfusion injury in all species tested so far, including humans. Although this phenomenon is known for more than 3 decades, the underlying signaling mechanisms are unclear in their details. Among various cardioprotective mediators, neuronal acetylcholine (ACh) and its signaling through cholinergic receptors is certainly involved. However, a non-neuronal cardiac cholinergic system (NNCCS) has been recently identified, suggesting a nervous system-independent, cardiomyocyte-derived ACh signaling with signal amplification by autocrine cholinergic receptor activation. Aim We aimed to examine the role of NNCCS-derived ACh as a mediator of cardioprotection. We therefore in a reductionist approach used isolated adult cardiomyocytes with hypoxic preconditioning (HPC). Methods Hearts from male Lewis rats were harvested, and adult ventricular cardiomyocytes were isolated. In suspended cardiomyocytes HPC was induced by one cycle of 10 min brief hypoxia and 20 min reoxygenation. Thirty min normoxia served as control. Afterwards, cardiomyocytes were exposed to 30 min sustained hypoxia and 5 min reoxygenation (H/R) or to 35 min normoxia as time control (TC). Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod-shaped, unstained (0.5% trypan blue) cells over all cells. In parallel experiments, physostigmine (0.2 mmol/L) was added to cardiomyocyte suspensions to block the ubiquitous ACh esterase. Intracellular ACh was quantified with and without HPC via liquid chromatography-coupled tandem mass spectrometry. In subsets, the nicotinic cholinergic receptor antagonists hexamethonium (1.0 µmol/L) and α-bungarotoxine (0.1 µmol/L) or the muscarinic cholinergic receptor antagonist atropine (0.1 µmol/L) were added to cardiomyocyte suspensions with and without HPC, respectively, and exposure to H/R or TC, respectively, afterwards. Again, cardiomyocyte viability was quantified. Results With HPC, the viability of cardiomyocytes subjected to H/R was better preserved than without (24±1% vs. 7±1%, figure A). Also, intracellular ACh was slightly (n.s.) increased after sustained hypoxia (6±2 pmol/mg protein), but significantly with HPC (13±3 pmol/mg protein) in comparison to baseline (3±1 pmol/mg protein) or TC (3±1 pmol/mg protein, figure B). However, this increase was only detectable in the presence of physostigmine (figure B). Addition of nicotinic and muscarinic cholinergic receptor antagonists attenuated the protection by HPC to the same extent (hexamethonium: 12±2%, α-bungarotoxine: 11±1%, atropine: 10±1%, figure A). Conclusion The NNCCS is activated by HPC and causal for HPC´s protection. HPC increases intracellular ACh, and cardiomyocyte protection is mediated via both nicotinic and muscarinic cholinergic receptors, suggesting an autocrine signal amplification on the cardiomyocyte level.

Protection of Mouse Cardiomyocytes by Plasma‐Dialysate from Volunteers Undergoing Remote Ischemic Conditioning Through STAT3 Signaling

Background Brief cycles of ischemia/reperfusion in a tissue or organ remote from the heart reduce myocardial infarct size (IS) in all species tested so far, including humans. Such remote ischemic conditioning (RIC) releases cardioprotective factors into the circulating blood. Plasma-dialysate from healthy volunteers undergoing RIC reduces IS in isolated perfused rodent hearts. Such protection is abrogated by the signal transducer and activator of transcription (STAT)3 inhibitor stattic. Whether or not such protection and its signaling are related to cardiomyocytes or other cellular components of the myocardium, is not established so far. Aim To characterize RIC's humoral protection on the cardiomyocyte level. Methods Healthy volunteers (4 males/ 4 females; 35±15 years) were subjected to RIC by 3 cycles of 5 min forearm ischemia/5 min reperfusion. Venous blood samples were taken before and 30 min after the RIC protocol, and a plasma-dialysate (12-14 kDa; dialysis tubing; dialysis over night at 4°C against normoxic buffer in a 1:10 volume ratio) was prepared. To confirm cardioprotection by humoral factors released by RIC, plasma-dialysate was infused into isolated rat hearts perfused at constant-pressure, before hearts were subjected to global-zero flow ischemia and reperfusion. IS was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infusion of plasma-dialysate after RIC protected, and IS was less after vs. before RIC: 20±6% vs. 39±9%. Ventricular mouse cardiomyocytes were isolated by enzymatic digestion and incubated with either normoxic buffer or plasma-dialysate for a 30 min baseline before being subjected to 50 min hypoxia and 5 min reoxygenation. Hypoxia was induced by exposing cardiomyocytes to iso-osmolar, glucose-free, pH 6.5 buffer. Reoxygenation was induced by normoxic hypo-osmolar (250 mOsm/L), glucose-containing (15 mmol/L) reoxygenation buffer. For time control, cardiomyocytes were incubated with normoxic buffer for 85 min. In subgroups, experiments were repeated under STAT3 blockade (stattic, 1 µmol/l). Cardiomyocyte viability was expressed as the percentage of rod-shaped, trypan-blue unstained cardiomyocytes over the total number of cells. Results Plasma-dialysate after RIC preserved viability better than plasma-dialysate before RIC (Figure). Incubation with stattic had no impact on cardiomyocyte viability per se, but abrogated the protection by plasma dialysate after RIC (Figure). Conclusion RIC´s humoral transfer of protection occurs at the cardiomyocyte level and causally involves STAT3.

Morphological differences in the pancreas of normo- and hypertensive rats after joint exposure of intermittent hypoxia and melatonin

The morphological changes in the pancreas of normo- and hypertensive rats after combined exposure to intermittent normobaric hypoxia and melatonin were studied. The study was conducted in the autumn on 24 normotensive (Wistar line) and 24 spontaneously hypertensive (SHR line) male rats. Experimental rats were daily exposed to hypoxic gas mixture in special sealed chamber. Hypoxic gas mixture (12 % oxygen in nitrogen) was supplied into this chamber from the membrane gas separator element in the intermittent mode: 15 min deoxygenation/ 15 min reoxygenation for 2 h. Exogenous melatonin was orally administered to experimental animals at 10 h in the morning at a dose of 5 mg / kg once a day. The total duration of the experiment was 28 d. Histological preparations of pancreatic tissue were prepared according to the standard methods. The morphometry of the preparations digital images was performed using the computer program ImageJ. The nature and severity of morphological changes in the pancreas of the experimental spontaneously hypertensive rats (hyperplasia of the nucleolus in the nucleus of exocrinocytes, a decrease in the amount of connective tissue) suggests that the combined effect of intermittent normobaric hypoxia and melatonin increases the activity of the exocrine part of the pancreas. At the same time, the activity of the endocrine part of the pancreas decreases, as evidenced by a decrease in the linear dimensions and area of the Langerhans islets, as well as the number of endocrinocytes contained in them. In Wistar rats, the combined effect of intermittent hypoxia and melatonin leads to the appearance of morphological signs of increased activity of both exocrine and endocrine pancreas functions. Thus, after prolonged combined exposure to intermittent normobaric hypoxia and melatonin, morphological signs of increased exocrine pancreas activity appear in both normotensive and hypertensive rats. At the same time, the activity of the endocrine function of the pancreas in experimental Wistar rats increases, while in SHR rats, on the contrary, decreases.

Alterations in cerebral and cardiac mitochondrial function in a porcine model of acute carbon monoxide poisoning

Objectives The purpose of this study is the development of a porcine model of carbon monoxide (CO) poisoning to investigate alterations in brain and heart mitochondrial function. Design Two group large animal model of CO poisoning. Setting Laboratory. Subjects Ten swine were divided into two groups: Control (n = 4) and CO (n = 6). Interventions Administration of a low dose of CO at 200 ppm to the CO group over 90 min followed by 30 min of re-oxygenation at room air. The Control group received room air for 120 min. Measurements Non-invasive optical monitoring was used to measure cerebral blood flow and oxygenation. Cerebral microdialysis was performed to obtain semi real time measurements of cerebral metabolic status. At the end of the exposure, both fresh brain (cortical and hippocampal tissue) and heart (apical tissue) were immediately harvested to measure mitochondrial respiration and reactive oxygen species (ROS) generation and blood was collected to assess plasma cytokine concentrations. Main results Animals in the CO group showed significantly decreased Complex IV-linked mitochondrial respiration in hippocampal and apical heart tissue but not cortical tissue. There also was a significant increase in mitochondrial ROS generation across all measured tissue types. The CO group showed a significantly higher cerebral lactate-to-pyruvate ratio. Both IL-8 and TNFα were significantly increased in the CO group compared with the Control group obtained from plasma. While not significant there was a trend to an increase in optically measured cerebral blood flow and hemoglobin concentration in the CO group. Conclusions Low-dose CO poisoning is associated with early mitochondrial disruption prior to an observable phenotype highlighting the important role of mitochondrial function in the pathology of CO poisoning. This may represent an important intervenable pathway for therapy and intervention.

Morphofunctional state of the lungs respiratory part in normotensive and hypertensive rats after combined exposure to intermittent hypoxia and melatonin

Aim. The purpose of this work was to study and compare the combined effect of intermittent normobaric hypoxia (INH) and melatonin on the morphological and biochemical indices of the lungs respiratory part in Wistar and spontaneous hypertensive rats (SHR). Material and methods. The studies were conducted on 48 young male rats Wistar and SHR lines. The experimental rats were daily exposed to hypoxic gas mixture (12 % oxygen in nitrogen) in intermittent mode: 15 min deoxygenation / 15 min reoxygenation for 2 hours. Animals also received oral melatonin at a dose of 5 mg /1 kg of body weight once daily.Results. The sizes of alveolus were reduced, but their number and placement density were increased in SHR rats after a combined effect of INH and melatonin. The results showed a significant decrease of the interalveolar septum thickness and a tendency to decrease of the hydroxyproline concentration in the lung tissue of the experimental rats, regardless of their line.Conclusions. The 28-day combined effect of INH and melatonin increase the total alveolar surface area and reduce the amount of connective tissue in the lungs of experimental rats. These changes can improve the efficiency of the intrapulmonary gas exchange processes.

Abstract 458: Chronic Inhibition of Protein Kinase C Improves Coronary Endothelial Function in the Setting of Diabetes and Hypoxia

Background: Both Diabetes (DM) and hypoxia/re-oxygenation (H/R) upregulate protein kinase C (PKC), resulting in coronary endothelial dysfunction. We hypothesized that chronic inhibition of PKC protects coronary endothelial function against DM and H/R injury. Methods: Genetically modified obesity and type-2 diabetic mice (female) were orally treated with or without selective PKCα/β inhibitor ruboxistaurin (10μg/g) for 4 weeks (n = 6/group). Coronary small arteries (70-110 μm in diameter) were then dissected from the harvested heart of mice. The isolated vessels were subjected to 60-min of hyperkalemic cardioplegic hypoxia (15°C) and then reperfused with oxygenated Krebs-Henseleit buffer for 60 minutes. At the end of 60-min re-oxygenation, all vessels was pretreated with endothelin-1 and then the responses to endothelium-dependent vasodilators, adenosine-diphosphate (ADP) and substance P were examined. Results: PKCα/β inhibition significantly improved the recovery of coronary endothelial function in the small coronary arteries of diabetic mice showing increased relaxation response to ADP and substance P in dose-dependent manner compared with the DM control (no-pretreatment alone) (*p<0.05). Conclusion: Chronic inhibition of PKC significantly improved the recovery of coronary endothelial function against a period of cardiolpegic H/R in the diabetic microvasculature. These data suggest that the PKC inhibitor ruboxistaurin represents a novel potential therapeutic for coronary endothelial dysfunctionin patients with DM following cardioplegic ischemia and reperfusion.

The combined effect of intermittent normobaric hypoxia and melatonin on the morphological changes of the spontaneously hypertensive rats pancreas

The aim of the study was to investigate the morphological changes in the pancreas of spontaneously hypertensive animals after the combined effect of intermittent normobaric hypoxia (INH) and melatonin.Materials and methods. The study was carried out in spring on 24 spontaneously hypertensive male rats (SHR line). The experimental animals were daily given a hypoxic gas mixture (12 % oxygen in nitrogen) in an intermittent mode: 15 min deoxygenation/15 min reoxygenation for 2 hours. The same rats were daily administered orally with exogenous melatonin at 10.00 at a dose of 5 mg/kg. The duration of the experiment was 28 days. Histological preparations were prepared according to a standard procedure. Morphometry of the digital images of preparations was performed using the computer program ImageJ.Results. Based on changes in morphometric parameters (reduction in the size of acini, exocrinocytes, height of the epithelium), it can be assumed that the combined effect of INH and melatonin reduces the activity of the exocrine function of the pancreas. In the endocrine part of the pancreas of experimental animals morphological signs of its activation were noted: the linear dimensions of the Langerhans islets, the number and the density of their endocrine cells increased. Experimental animals also showed a decrease in the width of interlobular and interacinar interlayers of connective tissue, which can be considered as a manifestation of the mechanism of structural adaptation that facilitates the transport of oxygen and nutrients to the parenchymal elements of the pancreas.Conclusions. The combined effect of intermittent normobaric hypoxia and melatonin leads to the appearance of morphological signs of a decrease in the activity of the exocrine part of the pancreas in hypertensive rats. At the same time, the activity of the endocrine gland function in these animals increased.

Морфологічні зміни щитоподібної залози спонтанно-гіпертензивних щурів після поєднаного впливу переривчастої нормобаричної гіпоксії та мелатоніну

The purpose of our work was to study the combined effects of intermittent normobaric hypoxia (INH) and melatonin on morphological changes of the thyroid gland of spontaneously hypertensive rats. The study was conducted on 24 spontaneously hypertensive (SHR lines) male rats in the spring period of the year. At the end of the experiment, the age of the animals was 4 months. Blood pressure in rats was determined at the caudal artery using a sphygmomanometer (S-2 “SHE” Germany). The rats were divided into 2 groups: I – control animals, and II – rats exposed to combined INH and melatonin. The experimental animals were daily given a hypoxic gas mixture (12 % O 2 ) in intermittent mode: 15 min deoxygenation/15 min reoxygenation during 2 hours. Another time of day (22 hours) the rats breathed air. Melatonin (“Unipharm Inc., USA”) was daily administered orally in a dose of 5 mg/kg at 10.00 a.m. The total duration of the experiment was 28 days. Histological preparations were prepared according to a standard methodic. Histomorphometry of the digital images of preparations was carried out using the computer program «Image J». It was found that 28-day combined effect of INH and melatonin has morphological signs of increasing the functional activity of the thyroid of spontaneously hypertensive rats. This is indicated by a decrease in the size of the follicles and colloid, an increase in the number of thyroid cells in the follicle, an increase in the follicular-colloidal index and a decrease in the colloid accumulation index, and a decrease in the width of the interfollicular connective tissue layers. These data may have not only a theoretical value, but also some practical interest in the use of hypoxic gas mixtures and melatonin to enhance the function of the thyroid gland in people suffering from arterial hypertension.

IHT‐induced neuronal protection is mediated by the microglia

Neuronal damage is the main cause of disability after various brain injuries, including ischemic stroke and traumatic brain injury. Recent years, it is being reported that microglia, a resident immune cell in the brain, shows dual edge roles. Intermittent hypoxic training (IHT) was introduced as a potential preventive or treatment program for the ischemic stroke and ethanol withdrawal syndrome. This study showed the effect of IHT on the function of microglia along with neuronal protection.MethodsEOC20, microglia, were randomly assigned to the following groups; 1) normoxic control, 2) oxygen‐glucose deprivation (OGD)‐reoxygenation, 3) IHT, and 4) IHT+ OGD‐reoxygenation. 3 days of IHT that consists of 5–8 daily and 5–10 min cycles of hypoxia with intervening 4 min of reoxygenation was applied to the microglia. OGD (0.1% O2, 90 min) and reoxygenation (24 hours) was used to simulate ischemic stroke and reperfusion. Additionally, the DMEM in which either normoxic or IHT‐treated microglia were grown was used to culture the neuronal cells. After word, the cell viability of neuronal cell exposed to the OGD and reoxygenation was measured. Calcein AM and the LDH assays have been used to compare the cell viability between groups. The microglia were characterized with staining CD45, 11b, CD68, and CD208 and by flow cytometry. Phagocytic activity of microglia was tested by using latex bead coated with FITC. ROS generation was also monitored by DCFDA assay. Both pro‐ and anti‐inflammatory cytokines were examined with commercial ELISA assay kits. Finally, the one‐way ANOVA followed by post hoc test was done to determine the statistical significance. The p‐value less than 0.05 was considered as a meaningful data.ResultsOGD and reoxygenation significantly decreased cell viability of microglia and interestingly, survived microglia from OGD and reoxygenation increased its phagocytic activity, which might benefit from the activated microglia. IHT was able to salvage the microglia from OGD and reoxygenation but also even more enhanced its phagocytic activity vs. OGD and reoxygenation group. This beneficial effect of IHT should be associated with increased antiinflammatory cytokine and shift of M1 to M2 stereotype. Last, it was observed that when neuronal cells were cultured in the DMEM in which microglia underwent 3 days of IHT, were able to be protected against OGD and reoxygenation. This might indicate humoral factors including cytokine released from microglia during IHT were an essential factor in maintaining cerebral circumstance, including neuron.ConclusionThis preliminary study suggests that microglia play a pivotal role in the neuronal damage in the ischemic stroke and can be a key target in developing ischemic stroke treatment. Furthermore, global intermittent hypoxic training might be potential and/or better intervention than an intervention that targets single cell type or organ in developing a treatment program for the ischemic stroke victims.Support or Funding InformationFaculty Development Grant, Tarleton State Univ Basic Research Program Grant, UNTHSCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of Acute Renal Injury After the Post-resuscitation Administration of Doxycycline in Surviving Newborn Piglets With Severe Hypoxia-Reoxygenation.

Background: Asphyxiated neonates often have myocardial dysfunction and renal insufficiency. Previously we demonstrated that doxycycline improved cardio-renal function through matrix metalloproteinase (MMP)-2 inhibition in an acute swine model of neonatal hypoxia-reoxygenation. The prolonged cardio-renal protective effects of doxycycline in neonates still remained unknown. We therefore hypothesized that the protective effects of doxycycline persisted in surviving subjects.Methods: Newborn piglets were instrumented and subjected to 1 h of hypoxia followed by reoxygenation with 21–25% oxygen and observed for 4 days. Intravenous doxycycline (30 mg/kg) or normal saline (1 mL, saline-control group) was given at 5 min of reoxygenation (n = 8/group) in a randomized, blinded fashion. Sham-operated piglets (n = 5) received no hypoxia-reoxygenation. At 96 h after reoxygenation, the left ventricular function was assessed by Millar® catheter. Renal injury was investigated by measuring plasma creatinine, urinary N-acetyl-D-glucosaminidase activity, renal tissue lactate and MMP-2 activity.Results: Both hypoxia-reoxygenation groups had similar hypoxic stress with severe lactate acidosis, and hemodynamic recovery. Doxycycline-treated piglets had higher urine output with lower urine N-acetyl-D-glucosaminidase, plasma creatinine, and renal MMP-2 activity (vs. saline-controls; all p < 0.05). These markers were all negatively correlated with urine output.Conclusions: In newborn piglets surviving hypoxia-reoxygenation, we observed a weak but significant and persistent attenuation of renal injury and improved recovery with the post-resuscitation administration of doxycycline.

Differences in vascular function between trained and untrained limbs assessed by near-infrared spectroscopy

The aim of this study was to examine whether differences in vascular responsiveness associated with training status would be more prominent in the trained limb (leg) than in the untrained limb(arm) microvasculature. Thirteen untrained (26 ± 5year) and twelve trained (29 ± 4year) healthy men were submitted to a vascular occlusion test (VOT) (2min baseline, 5min occlusion, and 8min re-oxygenation). The oxygen saturation signal (StO2) was assessed using near-infrared spectroscopy (NIRS) throughout the VOT. Vascular responsiveness within the microvasculature was evaluated by the re-oxygenation Slope 2 (Slope 2 StO2) and the area under the curve (StO2AUC) of (StO2) signal during re-oxygenation in the leg and arm. There was a significant interaction between training status and limb for the slope 2 StO2 (P < 0.01). The leg of the trained group showed a steeper slope 2 StO2 (1.35 ± 0.12%s-1) when compared to the slope 2 StO2 of the leg in their untrained counterparts (0.86 ± 0.09%s-1) (P < 0.05). There was a medium effect size of 0.58 for slope 2 StO2 on the arm and a large effect size of 1.21 for slope 2 StO2 on the leg. In addition, there was a small effect size of 0.24 for StO2AUC on the arm and a medium effect size of 0.64 for StO2AUC on the leg. The present study suggests that the vascular adaptations induced by lower limb endurance exercise training are more prominent in the trained limb than in the untrained limb microvasculature.