Influence of different oxygen modes on the blood oxygen transport and prooxidant-antioxidant status during hepatic ischemia/reperfusion

Oxygen supply was corrected in rabbits during the hepatic ischemia/reperfusion by means of different breathing mixtures: hypoxic (14.8 % O(2)+85.2 % N(2)), hyperoxic (78 % O(2)+20.2 % N(2)+ 1.8 % CO(2)), or hypercapnic (5 % CO(2) in air). Hepatic ischemia was induced for 30 min by ligation of hepatic artery, reperfusion period lasted 120 min. Indices of blood oxygen transport (p50(act), pCO(2), pH, pO(2), etc.) and prooxidant-antioxidant balance (Schiff bases, conjugated dienes, catalase, retinol, alpha-tocopherol) were measured in the blood and liver. The severity of reperfusion damage was evaluated by the activities of alanine and aspartate aminotransferases (ALT, AST) in the blood. Hepatic ischemia/reperfusion resulted in higher p50(act) in hepatic venous and mixed venous blood in all experimental groups. The changes of p50(act) were most marked in the hypercapnic group and were the weakest in the hypoxic group. The rise in p50(act) was accompanied by higher levels of lipid peroxidation products, ALT and AST in blood and liver homogenates, and by a simultaneous fall of alpha-tocopherol and retinol concentrations, except in the hypoxic group. Catalase activity at the end of reperfusion increased under normoxia, decreased under hyperoxia or hypercapnia and did not change under hypoxia. The moderate hypoxia during reperfusion was accompanied by a better balance between the mechanisms of reactive oxygen species production and inactivation that may be observed by optimal changes in p50act and reduced the hepatic damage in this pathological condition.     

Effect of sodium nitroprusside on hemoglobin oxygen binding properties of the blood during hepatic ischemia-reperfusion in rabbits

The parameters of blood oxygen transport were determined in rabbits during the hepatic ischemia/reperfusion (HIR) with or without sodium nitroprusside (SNP) administration. Hepatic ischemia was induced for 30 min by a. hepatica propria clamping, reperfusion lasted 120 min. Indices of blood oxygen transport (hemoglobin-oxygen affinity index (p50), pO2, pH, pCO2, HCO3-, TCO2, ABE, etc.) and nitrite/nitrate (NO(x)) amounts were measured in blood during HIR. Animals were subdivided into two groups: 1st group--HIR; 2nd--HIR plus SNP infusion (SNP, Sigma, i.v. 10 mcmol/kg). The experiments had shown that HIR led to significant acidic changes in the acid-base balance and high blood p50. The SNP infusion in the 2nd group led to less changing in the p50 values during HIR which were accompanied with high NO(x) levels. We conclude that oxyhemoglobin dissociation curve shift leftwards after SNP administration promotes the maintenance liver during ischemia-reperfusion.     

Molecular Mechanisms of Ischemia–Reperfusion Injury in Brain: Pivotal Role of the Mitochondrial Membrane Potential in Reactive Oxygen Species Generation

Stroke and circulatory arrest cause interferences in blood flow to the brain that result in considerable tissue damage. The primary method to reduce or prevent neurologic damage to patients suffering from brain ischemia is prompt restoration of blood flow to the ischemic tissue. However, paradoxically, restoration of blood flow causes additional damage and exacerbates neurocognitive deficits among patients who suffer a brain ischemic event. Mitochondria play a critical role in reperfusion injury by producing excessive reactive oxygen species (ROS) thereby damaging cellular components, and initiating cell death. In this review, we summarize our current understanding of the mechanisms of mitochondrial ROS generation during reperfusion, and specifically, the role the mitochondrial membrane potential plays in the pathology of cerebral ischemia/reperfusion. Additionally, we propose a temporal model of ROS generation in which posttranslational modifications of key oxidative phosphorylation (OxPhos) proteins caused by ischemia induce a hyperactive state upon reintroduction of oxygen. Hyperactive OxPhos generates high mitochondrial membrane potentials, a condition known to generate excessive ROS. Such a state would lead to a “burst” of ROS upon reperfusion, thereby causing structural and functional damage to the mitochondria and inducing cell death signaling that eventually culminate in tissue damage. Finally, we propose that strategies aimed at modulating this maladaptive hyperpolarization of the mitochondrial membrane potential may be a novel therapeutic intervention and present specific studies demonstrating the cytoprotective effect of this treatment modality.     

Nitric oxide and hemoglobin interactions in the vasculature.

As an endothelium-derived relaxing factor, nitric oxide (NO) maintains blood flow and O2 transport to tissues. Under normal conditions a delicate balance exists in the vascular system between endothelium-derived NO, an antioxidant, and the pro-oxidant elements of the vascular system, O-2, and peroxynitrite (a by-product of the reaction of NO and superoxide); in addition there is a balance between neurogenic tonic contraction and NO-mediated relaxation. The former balance can be disrupted in favor of peroxynitrite and hydrogen peroxide under the conditions of ischemia/reperfusion. This review suggests that NO may be beneficial, not only in terms of its new potential in improving O2 transport without accompanying significant increase in tissue blood flow, but also in its ability to suppress the prooxidative reagents of the vascular systems. These include NO-mediated inhibition of transendothelial migration by leukocyte and the antioxidative effects of NO with regard to ischemia/reperfusion; the relevance of these hypotheses to systemic administration of NO donors is discussed.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Effect of ozone treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion

This study investigates whether ozone could confer protection from hepatic ischemia reperfusion by modifying the accumulation of adenosine and xanthine during ischemia. A significant increase in both adenosine and xanthine accumulation was observed as a consequence of ATP degradation during hepatic ischemia. Adenosine exerts a protective effect on hepatic ischemia reperfusion injury since the elimination of endogenous adenosine accumulation with adenosine deaminase increased the hepatic injury associated with this process. On the other hand, the high xanthine levels observed after ischemia could exert deleterious effects during reperfusion due to reactive oxygen species generation from xanthine oxidase. The administration of allopurinol, an inhibitor of xanthine oxidase, attenuated the increase in reactive oxygen species and transaminase levels observed after hepatic reperfusion. Ozone treatment in liver maintained adenosine levels similar to those found after ischemia but led to a marked reduction in xanthine accumulation. In order to evaluate the role of both adenosine and xanthine, we tried to modify the protection confered by ozone, by modifying the concentrations of adenosine and xanthine. The metabolization of endogenous adenosine after ischemia abolished the protective effect conferred by ozone. When xanthine was administered previous to ozone treatment, the protection conferred by adenosine disappeared, showing both postischemic reactive oxygen species and transaminase levels similar to those found after hepatic ischemia reperfusion. Ozone would confer protection against the hepatic ischemia reperfusion injury by the accumulation of adenosine that in turns benefits the liver and by blocking the xanthine/xanthine oxidase pathway for reactive oxygen species generation.     

Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia

The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischema-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F₂-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60–250% increase of F₂-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90–320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 μmol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.     

A computational model of cardiomyocyte metabolism predicts unique reperfusion protocols capable of reducing cell damage during ischemia/reperfusion

If a coronary blood vessel is occluded and the neighboring cardiomyocytes deprived of oxygen, subsequent reperfusion of the ischemic tissue can lead to oxidative damage due to excessive generation of reactive oxygen species. Cardiomyocytes and their mitochondria are the main energy producers and consumers of the heart, and their metabolic changes during ischemia seem to be a key driver of reperfusion injury. Here, we hypothesized that tracking changes in cardiomyocyte metabolism, such as oxygen and ATP concentrations, would help in identifying points of metabolic failure during ischemia and reperfusion. To track some of these changes continuously from the onset of ischemia through reperfusion, we developed a system of differential equations representing the chemical reactions involved in the production and consumption of 67 molecular species. This model was validated and used to identify conditions present during periods of critical transition in ischemia and reperfusion that could lead to oxidative damage. These simulations identified a range of oxygen concentrations that lead to reverse mitochondrial electron transport at complex I of the respiratory chain and a spike in mitochondrial membrane potential, which are key suspects in the generation of reactive oxygen species at the onset of reperfusion. Our model predicts that a short initial reperfusion treatment with reduced oxygen content (5% of physiological levels) could reduce the cellular damage from both of these mechanisms. This model should serve as an open-source platform to test ideas for treatment of the ischemia reperfusion process by following the temporal evolution of molecular concentrations in the cardiomyocyte.     

Reduced inflammatory response and increased microcirculatory disturbances during hepatic ischemia-reperfusion injury in steatotic livers of ob/ob mice

Steatosis is a major risk factor for complications after liver surgery. Since neutrophil cytotoxicity is critical for ischemia-reperfusion injury in normal livers, the aim of the present study was to evaluate whether an exaggerated inflammatory response could cause the increased injury in steatotic livers. In C57Bl/6 mice, 60 min of warm hepatic ischemia triggered a gradual increase in hepatic neutrophil accumulation during reperfusion with peak levels of 100-fold over baseline at 12 h of reperfusion. Neutrophil extravasation and a specific neutrophil-induced oxidant stress (immunostaining for hypochlorous acid-modified epitopes) started at 6 h of reperfusion and peaked at 12-24 h. Ob/ob mice, which had a severe macrovesicular steatosis, suffered significantly higher injury (alanine transaminase activity: 18,000 +/- 2,100 U/l; 65% necrosis) compared with lean littermates (alanine transaminase activity: 4,900 +/- 720 U/l; 24% necrosis) at 6 h of reperfusion. However, 62% fewer neutrophils accumulated in steatotic livers. This correlated with an attenuated increase in mRNA levels of several proinflammatory genes in ob/ob mice during reperfusion. In contrast, sham-operated ob/ob mice had a 50% reduction in liver blood flow and 35% fewer functional sinusoids compared with lean littermates. These deficiencies in liver blood flow and the microcirculation were further aggravated only in ob/ob mice during reperfusion. The attenuated inflammatory response and reduced neutrophil-induced oxidant stress observed in steatotic livers during reperfusion cannot be responsible for the dramatically increased injury in ob/ob mice. In contrast, the aggravated injury appears to be mediated by ischemic necrosis due to massive impairment of blood and oxygen supply in the steatotic livers.     

Neutrophil elastase contributes to the development of ischemia-reperfusion-induced liver injury by decreasing endothelial production of prostacyclin in rats

We previously reported that nitric oxide (NO) derived from endothelial NO synthase (NOS) increased endothelial prostacyclin (PGI(2)) production in rats subjected to hepatic ischemia-reperfusion (I/R). The present study was undertaken to determine whether neutrophil elastase (NE) decreases endothelial production of PGI(2), thereby contributing to the development of I/R-induced liver injury by decreasing hepatic tissue blood flow in rats. Hepatic tissue levels of 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were transiently increased and peaked at 1 h after reperfusion, followed by a gradual decrease until 3 h after reperfusion. Sivelestat sodium hydrochloride and L-658,758, two NE inhibitors, reduced I/R-induced liver injury. These substances inhibited the decreases in hepatic tissue levels of 6-keto-PGF(1alpha) at 2 and 3 h after reperfusion but did not affect the levels at 1 h after reperfusion. These NE inhibitors significantly increased hepatic tissue blood flow from 1 to 3 h after reperfusion. Both hepatic I/R-induced increases in the accumulation of neutrophils and the microvascular permeability were inhibited by these two NE inhibitors. Protective effects induced by the two NE inhibitors were completely reversed by pretreatment with nitro-l-arginine methyl ester, an inhibitor of NOS, or indomethacin. Administration of iloprost, a stable derivative of PGI(2), produced effects similar to those induced by NE inhibitors. These observations strongly suggest that NE might play a critical role in the development of I/R-induced liver injury by decreasing endothelial production of NO and PGI(2), leading to a decrease in hepatic tissue blood flow resulting from inhibition of vasodilation and induction of activated neutrophil-induced microvascular injury.     

Protective Mechanisms of Hypothermia in Liver Surgery and Transplantation

Hepatic ischemia/reperfusion (I/R) injury is a side effect of major liver surgery that often cannot be avoided. Prolonged periods of ischemia put a metabolic strain on hepatocytes and limit the tolerable ischemia and preservation times during liver resection and transplantation, respectively. In both surgical settings, temporarily lowering the metabolic demand of the organ by reducing organ temperature effectively counteracts the negative consequences of an ischemic insult. Despite its routine use, the application of liver cooling is predicated on an incomplete understanding of the underlying protective mechanisms, which has limited a uniform and widespread implementation of liver-cooling techniques. This review therefore addresses how hypothermia-induced hypometabolism modulates hepatocyte metabolism during ischemia and thereby reduces hepatic I/R injury. The mechanisms underlying hypothermia-mediated reduction in energy expenditure during ischemia and the attenuation of mitochondrial production of reactive oxygen species during early reperfusion are described. It is further addressed how hypothermia suppresses the sterile hepatic I/R immune response and preserves the metabolic functionality of hepatocytes. Lastly, a summary of the clinical status quo of the use of liver cooling for liver resection and transplantation is provided.     

Myocardial fatty acid oxidation during ischemia and reperfusion

Inhibition of fatty acid oxidation is an early event in myocardial ischemia that most likely contributes to tissue injury by the accumulation of potentially toxic intermediates such as acylCoA and acylcarnitine. After reperfusion both myocardial oxygen consumption and fatty acid oxidation may rapidly recover to preischemic levels, even when contractile function remains depressed. The mechanisms underlying the apparent dissociation between contractile function and oxidative metabolism early during reperfusion are still controversial. In isolated rat hearts subjected to 60 min of no-flow ischemia myocardial oxygen consumption and oxidation of palmitate were lowered during reperfusion by 3 mM of NiCl₂ and by 6 µM of ruthenium red. The results provide indirect evidence for the hypothesis that intracellular calcium transport may be involved in the mechanisms responsible for the high oxidative metabolic rate early after reperfusion     

The influence of hepatic venous oxygen saturation on the liver's synthetic response to metabolic stress.

Hepatic oxygen consumption (HVO2) and hepatic venous oxygen saturation (ShvO2) were assessed in the isolated perfused rat liver under conditions that mimic critical illness in an effort to assess their utility in predicting the functional status of the liver. Flow rates were adjusted over the physiologic range of oxygen transport as indicated by the hepatic venous O2 saturation range of 10%-75%. HVO2 was found to be transport (HDO2) dependent only when perfusate conditions contained an increased counterregulatory hormone (glucagon, epinephrine, dexamethasone) stimulus or a high lactate concentration. In the absence of a metabolic load, (substrate and hormone-free perfusate), HVO2 was transport independent even at an ShvO2 as low as 10%. Although transport dependency of HVO2 is frequently used to infer tissue ischemia, hepatic oxygen consumption was poorly correlated with synthetic function under all conditions. In contrast, hepatic albumin production was directly related to ShvO2 at all levels of HDO2 and under all perfusion conditions indicating that this metabolic process is particularly sensitive to reductions in oxygen availability, which is more reliably predicted by venous saturation measurements. However, glucose and urea synthesis were almost independent of ShvO2. These findings indicate that various hepatic processes are affected differentially by stress conditions and flow alterations that may exist during critical illness, and protein synthesis is particularly sensitive to oxygen deprivation. Additionally, hepatic venous oxygen saturation measurement, but not HVO2, serves as a useful surrogate marker for hepatic albumin production suggesting that regional venous oximetry may play an important role in the detection of hepatic functional impairment.     

Oxygen radicals in lung pathology.

Pulmonary tissue can be damaged in different ways, for instance by xenobiotics (paraquat, butylated hydroxytoluene, bleomycin), during inflammation, ischemia reperfusion, or exposure to mineral dust or to normobaric pure oxygen levels. Reactive oxygen species are partly responsible for the observed pulmonary tissue damage. Several mechanisms leading to toxicity are described in this review. The reactive oxygen species induce bronchoconstriction, elevate mucus secretion, and cause microvascular leakage, which leads to edema formation. Reactive oxygen species even induce an autonomic imbalance between muscarinic receptor-mediated contraction and the beta-adrenergic-mediated relaxation of the pulmonary smooth muscle. Vitamin E and selenium have a regulatory role in this balance between these two receptor responses. The autonomic imbalance might be involved in the development of bronchial hyperresponsiveness, occurring in lung inflammation. Finally, several antioxidants are discussed which may be beneficial as therapeutics in several lung diseases.     

Pre‐conditions for eliminating mitochondrial dysfunction and maintaining liver function after hepatic ischaemia reperfusion

The liver, the largest organ with multiple synthesis and secretion functions in mammals, consists of hepatocytes and Kupffer, stem, endothelial, stellate and other parenchymal cells. Because of early and extensive contact with the external environment, hepatic ischaemia reperfusion (IR) may result in mitochondrial dysfunction, autophagy and apoptosis of cells and tissues under various pathological conditions. Because the liver requires a high oxygen supply to maintain normal detoxification and synthesis functions, it is extremely susceptible to ischaemia and subsequent reperfusion with blood. Consequently, hepatic IR leads to acute or chronic liver failure and significantly increases the total rate of morbidity and mortality through multiple regulatory mechanisms. An increasing number of studies indicate that mitochondrial structure and function are impaired after hepatic IR, but that the health of liver tissues or liver grafts can be effectively rescued by attenuation of mitochondrial dysfunction. In this review, we mainly focus on the subsequent therapeutic interventions related to the conservation of mitochondrial function involved in mitigating hepatic IR injury and the potential mechanisms of protection. Because mitochondria are abundant in liver tissue, clarification of the regulatory mechanisms between mitochondrial dysfunction and hepatic IR should shed light on clinical therapies for alleviating hepatic IR‐induced injury.     

A mathematical model of diffusional shunting of oxygen from arteries to veins in the kidney

To understand how arterial-to-venous (AV) oxygen shunting influences kidney oxygenation, a mathematical model of oxygen transport in the renal cortex was created. The model consists of a multiscale hierarchy of 11 countercurrent systems representing the various branch levels of the cortical vasculature. At each level, equations describing the reactive-advection-diffusion of oxygen are solved. Factors critical in renal oxygen transport incorporated into the model include the parallel geometry of arteries and veins and their respective sizes, variation in blood velocity in each vessel, oxygen transport (along the vessels, between the vessels and between vessel and parenchyma), nonlinear binding of oxygen to hemoglobin, and the consumption of oxygen by renal tissue. The model is calibrated using published measurements of cortical vascular geometry and microvascular Po(2). The model predicts that AV oxygen shunting is quantitatively significant and estimates how much kidney Vo(2) must change, in the face of altered renal blood flow, to maintain cortical tissue Po(2) at a stable level. It is demonstrated that oxygen shunting increases as renal Vo(2) or arterial Po(2) increases. Oxygen shunting also increases as renal blood flow is reduced within the physiological range or during mild hemodilution. In severe ischemia or anemia, or when kidney Vo(2) increases, AV oxygen shunting in proximal vascular elements may reduce the oxygen content of blood destined for the medullary circulation, thereby exacerbating the development of tissue hypoxia. That is, cortical ischemia could cause medullary hypoxia even when medullary perfusion is maintained. Cortical AV oxygen shunting limits the change in oxygen delivery to cortical tissue and stabilizes tissue Po(2) when arterial Po(2) changes, but renders the cortex and perhaps also the medulla susceptible to hypoxia when oxygen delivery falls or consumption increases.     

Improvement of rat liver function by energy repletion after the preservation period: implications for hepatic graft management

We very recently showed (using a blood-free perfusion model) that cold preservation sensitized rat hepatocyte functions to rewarming ischemic injury and that the injury can be prevented by repleting high-energy adenylates in the liver by short-term oxygenated warm reperfusion. Here we investigated whether short-term reperfusion after the preservation period can improve hepatic graft function in a blood reperfusion model. Eighteen-hour cold-preserved rat livers either untreated (Group A) or pretreated by 30-min oxygenated warm reperfusion after preservation (Group B) were subjected to 20-min ischemic rewarming and then reperfused with blood. Livers in Group B compared to Group A exhibited approx. three times increased bile production and bromosulfophthalein excretion, nearly 7-fold decreased swelling, and 1.2-fold improved blood flow. These results suggest that repletion of the energy by short-term oxygenated reperfusion after prolonged preservation may improve markedly initial hepatic graft function.     

Blood to brain transport after newborn cerebral ischemia/reperfusion injury

These experiments examine the transfer of urea, sodium, and sucrose from blood to brain in an animal model of newborn cerebral ischemia-reperfusion injury. Cerebral ischemia (20 min) was produced in anesthetized, ventilated piglets by increasing intracranial pressure above mean arterial blood pressure, thereby reducing cerebral perfusion pressure to zero. The blood to brain transfer of urea, sodium, and sucrose was then measured in sham control piglets and at 30 min and 2 hr of reperfusion following ischemia. At 30 min of reperfusion, urea and sodium transfer were increased while sucrose transfer was unchanged. However, at 2 hr of reperfusion, transfer of all three tracers was elevated. The difference in the time course of increased transport of these substances into the brain following ischemia cannot be explained by size differences, indicating that changes in the blood-brain barrier following ischemia are more complex than merely opening junctions between cells and creating leaky sites. Alterations in blood-brain barrier transport could participate in altered neuronal function after ischemia-reperfusion injury.     

Rheological of blood and oxygen transport in long-term adaptation to muscular load

The blood viscosity became reduced after a long-term muscular adaptation in dogs. The main adaptation mechanism is associated with an autoregulated haemodilution and improvement of the red blood cells' microrheology (deformities and aggregation). The findings suggest that reduction in the haemodilution and the blood oxygen capacity are accompanied by a heightened efficiency of the oxygen transport. A sufficient correlation exists between the blood fluidity parameters and the oxygen balance in the body. Value of the optimum haematocrit in oxygen transport, is discussed.