N-acetylcysteine effects on a murine model of chronic critical limb ischemia

During chronic limb ischemia, oxidative damage and inflammation are described. Besides oxidative damage, the decrease of tissue oxygen levels is followed by several adaptive responses. The purpose of this study was to determine whether supplementation with N-acetylcysteine (NAC) is effective in an animal model of chronic limb ischemia. Chronic limb ischemia was induced and animals were treated once a day for 30 consecutive days with NAC (30 mg/kg). After this time clinical scores were recorded and soleus muscle was isolated and lactate levels, oxidative damage and inflammatory parameters were determined. In addition, several mechanisms associated with hypoxia adaptation were measured (vascular endothelial growth factor - VEGF and hypoxia inducible factor - HIF levels, ex vivo oxygen consumption, markers of autophagy/mitophagy, and mitochondrial biogenesis). The adaptation to chronic ischemia in this model included an increase in muscle VEGF and HIF levels, and NAC was able to decrease VEGF, but not HIF levels. In addition, ex vivo oxygen consumption under hypoxia was increased in muscle from ischemic animals, and NAC was able to decrease this parameter. This effect was not mediated by a direct effect of NAC on oxygen consumption. Ischemia was followed by a significant increase in muscle myeloperoxidase activity, as well as interleukin-6 and thiobarbituric acid reactive substances species levels. Supplementation with NAC was able to attenuate inflammatory and oxidative damage parameters, and improve clinical scores. In conclusion, NAC treatment decreases oxidative damage and inflammation, and modulates oxygen consumption under hypoxic conditions in a model of chronic limb ischemia.     

间歇性低氧适应的心脏保护

间歇性低氧（intermittent hypoxia,IH）是指一定时间间断地暴露于低氧环境,而其余时间处于常氧环境。IH是机体某种生理和病理状态下的低氧形式。研究表明：间歇性低氧适应（IHadaptation）,类似缺血预适应（ischemic preconditioning,IPC）和长期高原低氧适应（long-termhigh-altitude hypoxic adaptation,LHA）,具有明显的心脏保护作用,表现为增强心肌对缺血／再灌注损伤的耐受性、限制心肌梗死面积和形态学改变、抗细胞凋亡、促进缺血／再灌注心脏舒缩功能的恢复,以及抗心律失常。尽管IH对心脏的保护作用不容质疑,但其作用机制远未阐明。IH心脏保护作用可能涉及氧的运输、能量代谢、神经体液调节、抗氧化酶、应激蛋白、腺苷系统、ATP敏感钾通道、线粒体及其钙调控、一氧化氮和蛋白激酶等多方面机制,并受低氧处理方式、动物年龄和性别等因素影响。IH心脏保护持续时间明显长于IPC,而对机体的不良影响远小于LHA,具有潜在的应用价值。     

Effects of ischemic and hypoxic hypoxia on VO2 and lactic acid output during tetanic contractions

We measured O2 uptake (VO2), CO2 output (VCO2), and net lactic acid output (L) during a 30-min period of repetitive 1/s isotonic tetanic contractions of the dog gastrocnemius-plantaris muscle group. The conditions were modest ischemic hypoxia (ischemia), hypoxia hypoxia (hypoxia), and free-flow normoxia (control). The major goal was to assess the effects of these perturbations on L during contractions. Ischemia and hypoxia were initiated just before the start of the contractions and at minute 7 of contractions in separate groups of experiments. Whenever applied, both ischemia and hypoxia reduced VO2 compared with the control values. When ischemia was initiated at the start of contractions, L was reduced transiently compared with the controls. When ischemia began at minute 7, L was increased modestly but transiently compared with the controls. When hypoxia was initiated at the start of contractions, L was increased during the entire period of contractions. The L pattern was the same as in the controls, rising to a maximal value at 3 min and declining steadily to a lower value at 30 min. When hypoxia began at minute 7, L declined initially at a slower rate than it did in the controls and was thereby elevated above the controls from 9 to 30 min. Ischemia was associated with a more rapid reduction in mechanical performance than hypoxia. The data suggest that the mechanisms of the decreased mechanical performance and VO2 are different for ischemia and hypoxia.     

Intermittent hypoxia attenuates ischemia／reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2／Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemiaJreperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts comparedwith normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reducemyocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.     

Effect of hypothermia on brain multi-parametric activities in normoxic and partially ischemic rats

Hypothermia, as well as anesthesia, are known to protect the brain against ischemia, hypoxia and other pathological damages. One of the mechanisms of this improvement could be by lowering brain function, and thereby lowering oxygen demand. We examined the effect of hypothermia on brain function and blood supply in awake and anesthetized rats and studied the interaction between partial ischemia and the responses to hypothermia. The brain function multiprobe (BFM) used enabled simultaneous measurements of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular K(+) concentration, DC potential and ECoG from the cerebral cortex in rats whose brain temperature was lowered by 5 degrees C. Hypothermia was induced in awake, anesthetized and brain ischemic-anesthetized rats. In anesthetized and ischemic-anesthetized rats, the time required for lowering the brain temperature by 5 degrees C was five times less than in the normal awake animals. No significant changes in CBF and NADH levels were found in response to hypothermia in the awake animals. In contrast, a significant decrease in extracellular K(+) concentration was recorded under hypothermia, probably due to the lower rate of depolarization. Hypothermia in anesthetized and in ischemic-anesthetized rats did not significantly affect the levels of mitochondrial NADH, CBF and extracellular K(+). Hypothermia under ischemia was expected to be more effective.     

Reactions of cellular structures of the brain of laboratory animals in oxygen deficiency

Analysis of the latest literature data and the authors' neuromorphological observations give the possibility to consider that structural reactions of the cerebral cellular elements of higher animals to oxygen insufficiency have a common base, but vary in dependence of the hypoxia form and the character of its influence and peculiarities of individual reactivity of the organism. Neurons and gliocytes possess rather effective mechanisms of resistance to hypoxia and ischemia, and are connected with intracellular regenerative and adaptive-compensatory processes. Hypoxic factor of pathogenesis of dystrophic-destructive changes in the cerebral cells is, however, subcoordinative, but not self-sufficing in all cases without any exception. Hypoxic and intoxicational lesions of the brain may have an independent cytological expression, despite the fact that differentiation of the combined signs is usually difficult.     

A novel long noncoding RNA FAF inhibits apoptosis via upregulating FGF9 through PI3K/AKT signaling pathway in ischemia–hypoxia cardiomyocytes

Long noncoding RNAs (lncRNAs) have been increasingly considered to play an important role in the pathological process of various cardiovascular diseases, which often bind to the proximal promoters of the protein-coding gene to regulate the protein expression. However, the functions and mechanisms of lncRNAs in cardiomyocytes have not been fully elucidated. High-throughput RNA sequencing was performed to identify the differently expressed lncRNAs and messenger RNAs (mRNAs) between acute myocardial infarction (AMI) rats and healthy controls. One novel lncRNA FGF9-associated factor (termed FAF) and mRNAs in AMI rats were verified by bioinformatics, real-time polymerase chain reaction or western blot. Moreover, RNA fluorescence in situ hybridization was performed to determine the location of lncRNA. Subsequently, a series of in vitro assays were used to observe the functions of lncRNA FAF in cardiomyocytes. The expression of lncRNA FAF and FGF9 were remarkably decreased in ischemia–hypoxia cardiomyocytes and heart tissues of AMI rats. Overexpression of FAF could significantly inhibit cardiomyocytes apoptosis induced by ischemia and hypoxia. Conversely, knockdown of lncRNA FAF could promote apoptosis in ischemia–hypoxia cardiomyocytes. Moreover, overexpression of lncRNA FAF could also increase the expression of FGF9. Knockdown of the FGF9 expression could promote apoptosis in cardiomyocytes with the insult of ischemia and hypoxia, which was consistent with the effect of lncRNA FAF overexpression on cardiomyocyte apoptosis. Mechanistically, FGF9 inhibited cardiomyocytes apoptosis through activating signaling tyrosine kinase FGFR2 via phosphoinositide 3-kinase/protein kinase B signaling pathway. Thus, lncRNA FAF plays a protective role in ischemia–hypoxia cardiomyocytes and may serve as a treatment target for AMI.     

Oxidative Stress, Hypoxia, and Ischemia-Like Conditions Increase the Release of Endogenous Amino Acids by Distinct Mechanisms in Cultured Retinal Cells

Abstract: The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe²⁺-induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca²⁺ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca²⁺-independent mechanism. The increased release observed in N -methyl- d -glucamine⁺ medium suggested the reversal of the Na⁺-dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na⁺-dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [³H]arachidonic acid and free radicals seems to affect the Na⁺-dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca²⁺]_i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism.     

Comparison of two oxygen delivery systems for safe sedation and spinal anesthesia in sheep

Proper oxygenation is critical to prevent hypoxia and myocardial ischemia in animals during pharmacological sedation. The authors compared the efficacy of two oxygen delivery masks during sedation and spinal anesthesia for knee surgery in sheep.     

Effects of adaptation to stress exposure and periodic hypoxia on bioelectric activity of cardiomyocytes of isolated heart in ischemia and reperfusion]

Action potential (AP) of cardiomyocytes was recorded in experiments on isolated perfused according to Langendorf rat hearts. The effect was estimated of preliminary adaptation to intermittent hypobaric hypoxia or to repeated short-term stress exposure on the resting potential (RP) and the amplitude and duration of action potential (APD) in global ischemia and reperfusion. It was shown that adaptation to hypoxia is more effective in prevention of ischemic fall of RP, AP and APD. In reperfusion, the parameters enumerated restored more quickly and efficiently in hearts from adapted to stress animals.     

DNA fragmentation during exposure of rat cerebella to ethanol under hypoxia imposed in vitro

To gain a better understanding into the mechanisms of damage incurred by neurons in periods following heavy alcohol exposure during development, we used an in vitro system to monitor the effects of alcohol and hypoxia on cell survival and DNA integrity. Samples representing the first few hours of exposure to alcohol and hypoxia were compared to those resulting from hypoxia alone. Measurements were taken from cell counts using Trypan blue exclusion and TUNEL assays as well as digital scans of the ethidium bromide fluorescence of genomic DNA isolated from the treated tissue. We found that DNA degradation from hypoxia was accelerated by several hours in the presence of 100 mM ethanol. This result depended on age, with adult animals (>8 months) having a similar response to 4-day postnatal animals, while the effect on 10-day postnatal animals and those of intermediate age (45 days postnatal) was increasingly delayed. Different methods of inducing the processive degradation of DNA produced laddering typical of apoptosis, a biphasic degradative process, or patterns usually associated with necrosis.     

MCC-134, a blocker of mitochondrial and opener of sarcolemmal ATP-sensitive K+ channels, abrogates cardioprotective effects of chronic hypoxia

We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.     

Effects of adaptation to periodic hypoxia on bioelectric activity of cardiomyocytes of isolated heart in ischemia and reperfusion

Experiments on isolated Wistar rat heart perfused according to Langendorff showed that preliminary adaptation of rats to intermittent hypobaric hypoxia limited the fall of values of the resting potential and the amplitude and duration of action potential characteristic for ischemia. Under similar conditions, adaptation considerably reduced the increased time of impulse conduction along the myocardium. In reperfusion, the parameters enumerated restored much more efficiently in hearts from adapted animals than in controls. The role of these changes in the antiarrhythmic effect of adaptation to intermittent hypoxia is under discussion.     

Comparative assessment of the effect of adaptation to stress exposure and high altitude hypoxia on heart resistance to reperfusion injury after total ischemia]

Experiments on isolated Wistar rat hearts perfused according to Langendorff showed that adaptation to stress exposure limited the depression of contraction amplitude and contracture and possessed an antiarrhythmic effect in reperfusion. Furthermore, adaptation to stress exposure efficiently limited reperfusion damage to sarcolemma. It was shown that adaptation to hypoxia did not result in any increase in the heart resistance to reperfusion damage following total ischemia. Possible mechanisms of differences in the protective effects of adaptation to stress exposure and hypoxia are discussed.     

Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway

Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2beta, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3'-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion.     

Influence of melatonin pretreatment and preconditioning by hypobaric hypoxia on the development of cortical photothrombotic ischemic lesion

Photothrombotic model of ischemia (PT) is based on free radical-mediated endothelial dysfunction followed by thrombosis. Free radicals are also involved in hypoxic preconditioning. We tested the sensitivity of PT to preconditioning with hypobaric hypoxia and to pretreatment with melatonin. In adult Wistar rats, after intravenous application of Rose Bengal, a stereo-tactically defined spot on the denuded skull was irradiated by a laser for 9 min. The first experimental group underwent hypobaric hypoxia three days before irradiation. In the second experimental group, melatonin was applied intraperitoneally one hour before irradiation. Three days after irradiation, animals were sacrificed, the brains perfused, and stained with TTC. Ischemic lesions were divided into grades (I, II, III). In the control group (where no manipulation preceded photothrombosis), most animals displayed deep damage involving the striatum (grade III). The group pre-exposed to hypoxia showed similar results. Only 28.57 % of the melatonin pretreated animals exhibited grade III lesions, and in 57.14 % no signs of lesions were detected. Pre-exposure to hypoxia was not protective in our model. Pretreatment with melatonin lead to a significant reduction of the number of large ischemic lesions. This result is probably caused by protection of endothelial cells by melatonin.     

Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia

Hypoxia is one of the most frequently encountered stresses in health and disease. The duration, frequency, and severity of hypoxic episodes are critical factors determining whether hypoxia is beneficial or harmful. Adaptation to intermittent hypoxia has been demonstrated to confer cardiovascular protection against more severe and sustained hypoxia, and, moreover, to protect against other stresses, including ischemia. Thus, the direct and cross protective effects of adaptation to intermittent hypoxia have been used for treatment and prevention of a variety of diseases and to increase efficiency of exercise training. Evidence is mounting that nitric oxide (NO) plays a central role in these adaptive mechanisms. NO-dependent protective mechanisms activated by intermittent hypoxia include stimulation of NO synthesis as well as restriction of NO overproduction. In addition, alternative, nonenzymic sources of NO and negative feedback of NO synthesis are important factors in optimizing NO concentrations. The adaptive enhancement of NO synthesis and/or availability activates or increases expression of other protective factors, including heat shock proteins, antioxidants and prostaglandins, making the protection more robust and sustained. Understanding the role of NO in mechanisms of adaptation to hypoxia will support development of therapies to prevent and treat hypoxic or ischemic damage to organs and cells and to increase adaptive capabilities of the organism.     

缩醛基毛冬青提取化合物R4对缺血缺氧心肌的保护作用

目的：研究缩醛基毛冬青提取化合物R4对缺血缺氧心肌的保护作用,以便为缩醛基毛冬青提取化合物R4的临床新用途提供实验依据。方法：采用小鼠常压耐缺氧实验、夹闭气管小鼠心电消失时间、垂体后叶素所致大鼠心肌缺血模型及大鼠冠脉结扎所致的心肌缺血模型,观察缩醛基毛冬青提取化合物R4对缺血缺氧心肌的保护作用。结果：缩醛基毛冬青提取化合物R4（1.0、2.0、4.0 mg/kg）均能显著延长小鼠常压耐缺氧条件下的存活时间,延长夹闭气管小鼠心电消失时间,缩醛基毛冬青提取化合物R4（0.75、1.5和3.0 mg/kg）能拮抗垂体后叶素引起的心电图变化,并能明显降低结扎冠脉所致大鼠的心肌梗塞范围。结论：缩醛基毛冬青提取化合物R4对缺血缺氧心肌具有明显保护作用,其效应与剂量呈相关性,其机制可能是通过扩张冠脉,增加心肌的供血供氧而发挥抗心肌缺血的作用。