Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex

Isoprostanes, neuroprostanes, isofurans, and neurofurans have all become attractive biomarkers of oxidative damage and lipid peroxidation in brain tissue. Asphyxia and subsequent reoxygenation cause a burst of oxygen free radicals. Isoprostanes and isofurans are generated by free radical attacks of esterified arachidonic acid. Neuroprostanes and neurofurans are derived from the peroxidation of docosahexanoic acid, which is abundant in neurons and could therefore more selectively represent oxidative brain injury. Newborn piglets (age 12-36h) underwent hypoxia until the base excess reached -20mmol/L or the mean arterial blood pressure dropped below 15mm Hg. They were randomly assigned to receive resuscitation with 21, 40, or 100% oxygen for 30min and then ventilation with air. The levels of isoprostanes, isofurans, neuroprostanes, and neurofurans were determined in brain tissue (ng/g) isolated from the prefrontal cortex using gas chromatography-mass spectrometry (GC/MS) with negative ion chemical ionization (NICI) techniques. A control group underwent the same procedures and observations but was not submitted to hypoxia or hyperoxia. Hypoxia and reoxygenation significantly increased the levels of isoprostanes, isofurans, neuroprostanes, and neurofurans in the cerebral cortex. Nine hours after resuscitation with 100% oxygen for 30min, there was nearly a 4-fold increase in the levels of isoprostanes and isofurans compared to the control group (P=0.007 and P=0.001) and more than a 2-fold increase in neuroprostane levels (P=0.002). The levels of neuroprostanes and neurofurans were significantly higher in the piglets that were resuscitated with supplementary oxygen (40 and 100%) compared to the group treated with air (21%). The significance levels of the observed differences in neuroprostanes for the 21% vs 40% comparison and the 21% vs 100% comparison were P<0.001 and P=0.001, respectively. For neurofurans, the P values of the 21% vs 40% comparison and the 21% vs 100% comparison were P=0.036 and P=0.025, respectively. Supplementary oxygen used for the resuscitation of newborns increases lipid peroxidation in brain cortical neurons, a result that is indicative of oxidative brain damage. These novel findings provide new knowledge regarding the relationships between oxidative brain injury and resuscitation with oxygen.     

Proteomic analysis of cardiac metabolic enzymes in asphyxiated newborn piglets

Hypoxia/reoxygenation (H/R) creates an energetic deficiency in the heart, which may contribute to myocardial dysfunction. We hypothesized that H/R-induced impairment of cardioenergetic enzymes occurs in asphyxiated newborn animals. After hypoxia for 2 h (10-15% oxygen), newborn piglets were resuscitated with 100% oxygen for 1 h, followed by 21% oxygen for 3 h. Sham-operated control piglets had no H/R. Hemodynamic parameters in the piglets were continuously measured. At the end of experiment, hearts were isolated for proteomic analysis. In asphyxiated hearts, the level of isocitrate dehydrogenase and malate dehydrogenase was reduced compared to controls. Inverse correlations between the level of myocardial malate dehydrogenase and cardiac function were observed in the control, but not the H/R hearts. We conclude that reoxygenation of asphyxiated newborn piglets reduces the level of myocardial isocitrate dehydrogenase and malate dehydrogenase. While the cause is not clear, it may be related to the impaired tricarboxylic acid cycle pathway and energy production in the heart.     

Comparison of Postasphyxial Resuscitation with 100% and 21% Oxygen on Cortical Oxygen Pressure and Striatal Dopamine Metabolism in Newborn Piglets

Abstract: The present study tests the hypothesis that ventilation with 100% O₂ during recovery from asphyxia leads to greater disturbance in brain function, as measured by dopamine metabolism, than does ventilation with 21% oxygen. This hypothesis was tested using mechanically ventilated, anesthetized newborn piglets as an animal model. Cortical oxygen pressure was measured by the oxygen-dependent quenching of phosphorescence, striatal blood flow by laser Doppler, and the extracellular levels of dopamine and its metabolites by in vivo microdialysis. After establishment of a baseline, both the fraction of inspired oxygen (FiO₂) and the ventilator rate were reduced in a stepwise fashion every 20 min over a 1-h period. For the subsequent 2-h recovery, the animals were randomized to breathing 21 or 100% oxygen. It was observed that during asphyxia cortical oxygen pressure decreased from 36 to 7 torr, extracellular dopamine increased 8,300%, and dihydroxyphenylacetic acid and homovanillic acid decreased by 65 and 60%, respectively, compared with controls. During reoxygenation after asphyxia, cortical oxygen pressure was significantly higher in the piglets ventilated with 100% oxygen than in those ventilated with 21% oxygen (19 vs. 11 torr). During the first hour of reoxygenation, extracellular dopamine levels decreased to ～200% of control in the 21% oxygen group, whereas these levels were still much higher in the 100% oxygen group (～500% of control). After ～2 h of reoxygenation, there was a secondary increase in extracellular dopamine to ～750 and ～3,000% of baseline for the animals ventilated with 21 and 100%, respectively. It is concluded that although 100% FiO₂ after asphyxia increases cortical oxygenation compared with 21% FiO₂, it also results in poorer recovery in dopamine metabolism and higher secondary release of striatal dopamine. The resulting increased extracellular levels of dopamine may exacerbate posthypoxic cerebral injury.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.     

Relationships of Dopamine, Cortical Oxygen Pressure, and Hydroxyl Radicals in Brain of Newborn Piglets During Hypoxia and Posthypoxic Recovery

Abstract: The present study describes the relationships of extracellular striatal dopamine, cortical oxygen pressure, and striatal hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic reoxygenation. Hypoxia was induced by reducing the fraction of inspired oxygen (F_iO₂) from 22% (control) to 7% for 1 h. The F_iO₂ was then returned to the control value and measurements were continued for 2 h. Cerebral oxygen pressure was measured by the oxygen dependent quenching of phosphorescence and extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and hydroxy radicals in the striatum were determined by in vivo microdialysis. Hypoxia decreased the cortical oxygen pressure from 47 ± 2 to 9 ± 1.3 torr (p < 0.001); the levels of extracellular dopamine in the striatum increased to 16,000 ± 3,270% of control (p < 0.01), whereas the levels of DOPAC and HVA decreased to 25.3 ± 6% (p < 0.001) and 36 ± 5% (p < 0.01) of control, respectively. Compared with control, the hydroxyl radical levels at each time point were not significantly increased during hypoxia, although the sum of the measured values was significantly increased (p < 0.05). During the first 5 min after F_iO₂ was returned to 22%, the cortical oxygen pressure increased to control values and stayed at this level for the remainder of the measurement period. The extracellular level of dopamine declined to values not statistically different from control during 40 min of reoxygenation. During the first 10 min of reoxygenation, DOPAC and HVA further decreased and then began to slowly increase. By 70 min of reoxygenation, the values were not significantly different from control. Hydroxyl radicals were above control during the entire period of reoxygenation, with maximal values observed after 100 min of reoxygenation. This increase was largely abolished by injecting the animals with α-methyl-p-tyrosine 5 h before hypoxia, a procedure that depleted the brain of dopamine. Our results suggest that oxidation of striatal dopamine during posthypoxic reoxygenation is at least partly responsible for the observed increase in striatal level of hydroxyl radicals that may exacerbate posthypoxic cerebral injury.     

Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

Twotypesofcellulardemisecanoccursimultaneouslyintissuesorculturedcellbynecrosisandapoptosis.Lossofmembraneintegrity,celledemaandbreak,andthecellcomponentsre-leasedoutarethecharacteristicsofnecrosis.Whilethecellapoptosisisaprogramcelldeathcodedbygeneandactivatedseriousendogenousenzymes[1].Recentstudieshavedemonstratedthatmyocardialischemia-reperfusioninjuryresultedinapoptoticcelldeathinadditiontotissuenecrosis[2—4].Oxygenstressisoneofthereasonsthatcausedcellapoptosisandtheoxygenradicalsinthest…     

Effects of post-resuscitation treatment with N-acetylcysteine on cardiac recovery in hypoxic newborn piglets

Aims

Although N-acetylcysteine (NAC) can decrease reactive oxygen species and improve myocardial recovery after ischemia/hypoxia in various acute animal models, little is known regarding its long-term effect in neonatal subjects. We investigated whether NAC provides prolonged protective effect on hemodynamics and oxidative stress using a surviving swine model of neonatal asphyxia.

Methods and Results

Newborn piglets were anesthetized and acutely instrumented for measurement of systemic hemodynamics and oxygen transport. Animals were block-randomized into a sham-operated group (without hypoxia-reoxygenation [H–R, n = 6]) and two H-R groups (2 h normocapnic alveolar hypoxia followed by 48 h reoxygenation, n = 8/group). All piglets were acidotic and in cardiogenic shock after hypoxia. At 5 min after reoxygenation, piglets were given either saline or NAC (intravenous 150 mg/kg bolus + 20 mg/kg/h infusion) via for 24 h in a blinded, randomized fashion. Both cardiac index and stroke volume of H-R controls remained lower than the pre-hypoxic values throughout recovery. Treating the piglets with NAC significantly improved cardiac index, stroke volume and systemic oxygen delivery to levels not different from those of sham-operated piglets. Accompanied with the hemodynamic improvement, NAC-treated piglets had significantly lower plasma cardiac troponin-I, myocardial lipid hydroperoxides, activated caspase-3 and lactate levels (vs. H-R controls). The change in cardiac index after H-R correlated with myocardial lipid hydroperoxides, caspase-3 and lactate levels (all p<0.05).

Conclusions

Post-resuscitation administration of NAC reduces myocardial oxidative stress and caused a prolonged improvement in cardiac function and in newborn piglets with H-R insults.     

活性氧和线粒体ATP敏感钾通道介导肿瘤坏死因子α对缺氧/复氧心肌的保护作用

在培养的乳鼠心肌细胞上,研究肿瘤坏死因子α(TNF-α)对缺氧/复氧损伤心肌的保护作用的机制。结果发现：(1)用TNF-α(10—500U／ml)预处理,缺氧/复氧后心肌细胞内锰超氧化物歧化酶(Mn-SOD)活性增高、乳酸脱氢酶(LDH)释放量减少(P＜0．05);(2)用抗氧化剂N-乙酰半既氨酸(NAC,1mmol/L)、抗霉素A(antimycin A,50μmol/L)、2-巯基丙酰氨基乙酸(2-MPG,400μmol/L)和铜／锌超氧化物歧化酸(Cu/Zn,SOD)抑制剂二乙基二硫代氨基甲酸盐(DDC,100nmol/L)预处理,可取消TNF-α的抑制缺氧/复氧心肌细胞LDH释放和诱导Mn-SOD活性增高的作用;(3)mitoKATP通道抑制剂5-羟基癸酸(5-HD)预处理可阻断TNF-α对缺氧/复氧心肌细胞的保护作用;选择性mitoKATP通道开放剂diazoxide(50μmol/L)预处理可减少复氧后心肌细胞LDH的释放(P＜0．01),其作用可被5-HD(100μmol/L)和NAC所抑制。上述结果表明,活性氧和线粒体ATP敏感钾通道参与介导TNF-α对缺氧/复氧损伤的心肌保护作用。     

Angiostatin is negatively associated with coronary collateral growth in patients with coronary artery disease

Angiostatin, an inhibitor of tumor angiogenesis, is produced by the actions of matrix metalloproteinases (MMP) on plasminogen. Recently, we reported that angiostatin levels are increased in a model of inadequate coronary collateral growth and angiogenesis in response to ischemia, despite high levels of vascular endothelial growth factor (VEGF). We hypothesized that angiostatin levels are negatively associated with collateral formation in patients. Coronary angiograms from 37 patients undergoing coronary bypass surgery were evaluated for the absence of angiographically visible collaterals (Rentrop scores of 0) or the presence of Rentrop classification grade 3 (well developed) collaterals. Pericardial fluid was obtained from each patient during the bypass procedure, and the sample was analyzed for angiostatin, plasminogen, and VEGF (Western analysis) and for combined activities of MMP-2 and MMP-9 (zymographic analysis). In patients with no collaterals, angiostatin level was greater compared with that in patients with well-developed collaterals (3.1 +/- 0.2 vs. 2.3 +/- 0.1 optical density units, P < 0.05). Neither MMP activities nor VEGF levels were different between the two groups of patients. The higher levels of angiostatin in patients with no visible collaterals were reflective of a higher concentration of plasmin/plasminogen (6.2 +/- 0.7 vs. 4.2 +/- 0.5 optical density units, P < 0.05) compared with those in patients with well-developed collateral vessels. Our results support the concept that the growth inhibitor angiostatin may have a negative impact on coronary collateral growth in patients. Perhaps therapies attempting to provoke coronary collateral growth should incorporate approaches to limit or neutralize the effects of growth inhibitors.     

p21 WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.     

Opticin production is reduced by hypoxia and VEGF in human retinal pigment epithelium via MMP-2 activation

Opticin, a small leucine rich repeat protein (SLRP) contributes to vitreoretinal adhesion. This study was conducted to investigate the effects of hypoxia and vascular endothelial growth factor (VEGF) on matrix metalloproteinase (MMP) mediated opticin production in retinal pigment epithelium (RPE) cells. Primary cultured human RPE cells were treated with hypoxia (low oxygen and cobalt chloride) or VEGF (0-100 ng/mL). The mRNA levels of opticin and the protein levels of intra and extracellular opticin in RPE cells were examined by RT-PCR and Western blot assay, respectively. Furthermore, the MMP activity was analyzed by zymography, and EDTA was used as an MMP inhibitor. Analysis of the effect of MMP-2 on opticin was performed by recombinant human (rh) MMP-2 stimulation in RPE cultures and by human vitreous sample digestion with activated rhMMP-2. Our results showed that opticin was expressed by primary cultured human RPE cells. Hypoxia and VEGF stimulation did not alter opticin mRNA and protein expression in RPE cells, but markedly decreased the protein levels of extracellular opticin following increased latent MMP-2 activity. The VEGF- and hypoxia induced opticin degradation in the culture medium was blocked by EDTA. Together, opticin levels in the culture medium were also reduced after rhMMP-2 treatment. In addition, opticin in human vitreous samples could be cleaved by rhMMP-2. These results reveal that VEGF and hypoxia could decrease opticin protein levels in the human RPE secretome, and that opticin may be an enzymatic substrate for MMP-2.     

Cycling hypoxia up-regulates thioredoxin levels in human MDA-MB-231 breast cancer cells

The thioredoxin system is a key cellular antioxidant system and is highly expressed in cancer cells, especially in more aggressive and therapeutic resistant tumors. We analysed the expression of the thioredoxin system in the MDA-MB-231 breast cancer cell line under conditions mimicking the tumor oxygen microenvironment. We grew breast cancer cells in either prolonged hypoxia or hypoxia followed by various lengths of reoxygenation and in each case cells were cultured with or without a hypoxic cycling preconditioning (PC) phase preceding the hypoxic growth. Flow cytometry-based assays were used to measure reactive oxygen species (ROS) levels. Cells grown in hypoxia showed a significant decrease in ROS levels compared to normoxic cells, while a significant increase in ROS levels over normoxic cells was observed after 4 h of reoxygenation. The PC pre-treatment did not have a significant effect on ROS levels. Thioredoxin levels were also highest after 4 h of reoxygenation, however cells subjected to PC pre-treatment displayed even higher thioredoxin levels. The high level of intracellular thioredoxin was also reflected on the cell surface. Reporter assays showed that activity of the thioredoxin and thioredoxin reductase gene promoters was also highest in the reoxygenation phase, although PC pre-treatment did not result in a significant increase over non-PC treated cells. The use of a dominant negative Nrf-2 negated the increased thioredoxin promoter activity during reoxygenation. This data suggests that the high levels of thioredoxin observed in tumors may arise due to cycling between hypoxia and reoxygenation.     

Metabolomic Analyses of Plasma Reveals New Insights into Asphyxia and Resuscitation in Pigs

Background

Currently, a limited range of biochemical tests for hypoxia are in clinical use. Early diagnostic and functional biomarkers that mirror cellular metabolism and recovery during resuscitation are lacking. We hypothesized that the quantification of metabolites after hypoxia and resuscitation would enable the detection of markers of hypoxia as well as markers enabling the monitoring and evaluation of resuscitation strategies.

Methods and Findings

Hypoxemia of different durations was induced in newborn piglets before randomization for resuscitation with 21% or 100% oxygen for 15 min or prolonged hyperoxia. Metabolites were measured in plasma taken before and after hypoxia as well as after resuscitation. Lactate, pH and base deficit did not correlate with the duration of hypoxia. In contrast to these, we detected the ratios of alanine to branched chained amino acids (Ala/BCAA; R².adj = 0.58, q-value<0.001) and of glycine to BCAA (Gly/BCAA; R².adj = 0.45, q-value<0.005), which were highly correlated with the duration of hypoxia. Combinations of metabolites and ratios increased the correlation to R²adjust = 0.92. Reoxygenation with 100% oxygen delayed cellular metabolic recovery. Reoxygenation with different concentrations of oxygen reduced lactate levels to a similar extent. In contrast, metabolites of the Krebs cycle (which is directly linked to mitochondrial function) including alpha keto-glutarate, succinate and fumarate were significantly reduced at different rates depending on the resuscitation, showing a delay in recovery in the 100% reoxygenation groups. Additional metabolites showing different responses to reoxygenation include oxysterols and acylcarnitines (n = 8–11, q<0.001).

Conclusions

This study provides a novel strategy and set of biomarkers. It provides biochemical in vivo data that resuscitation with 100% oxygen delays cellular recovery. In addition, the oxysterol increase raises concerns about the safety of 100% O₂ resuscitation. Our biomarkers can be used in a broad clinical setting for evaluation or the prediction of damage in conditions associated with low tissue oxygenation in both infancy and adulthood. These findings have to be validated in human trials.     

Inosine, not adenosine, initiates endothelial glycocalyx degradation in cardiac ischemia and hypoxia

Ischemia/reperfusion and hypoxia/reoxygenation of the heart both induce shedding of the coronary endothelial glycocalyx. The processes leading from an oxygen deficit to shedding are unknown. An involvement of resident perivascular cardiac mast cells has been proposed. We hypothesized that either adenosine or inosine or both, generated by nucleotide catabolism, attain the concentrations in the interstitial space sufficient to stimulate A3 receptors of mast cells during both myocardial ischemia/reperfusion and hypoxia/reoxygenation. Isolated hearts of guinea pigs were subjected to either normoxic perfusion (hemoglobin-free Krebs-Henseleit buffer equilibrated with 95% oxygen), 20 minutes hypoxic perfusion (buffer equilibrated with 21% oxygen) followed by 20 minutes reoxygenation, or 20 minutes stopped-flow ischemia followed by 20 minutes normoxic reperfusion (n = 7 each). Coronary venous effluent was collected separately from so-called transudate, a mixture of interstitial fluid and lymphatic fluid appearing on the epicardial surface. Adenosine and inosine were determined in both fluid compartments using high-performance liquid chromatography. Damage to the glycocalyx was evident after ischemia/reperfusion and hypoxia/reoxygenation. Adenosine concentrations rose to a level of 1 μM in coronary effluent during hypoxic perfusion, but remained one order of magnitude lower in the interstitial fluid. There was only a small rise in the level during postischemic perfusion. In contrast, inosine peaked at over 10 μM in interstitial fluid during hypoxia and also during reperfusion, while effluent levels remained relatively unchanged at lower levels. We conclude that only inosine attains levels in the interstitial fluid of hypoxic and postischemic hearts that are sufficient to explain the activation of mast cells via stimulation of A3-type receptors.     

Effects of hypoxia and reoxygenation on adult rat heart cell metabolism

Isolated adult rat heart cells were used to study the effects of oxygen deprivation followed by reoxygenation upon myocardial metabolism. Calcium-tolerant nonbeating myocytes were incubated for 5, 30, or 60 min under 100% oxygen or 100% nitrogen and then rinsed with oxygenated buffer. Substrate oxidation was studied by incubating the cells with 14C-labeled glucose, pyruvate, or octanoate and determining the rates of 14CO2 production from the individual substrates. After 5 min of hypoxia, metabolism of glucose, as assessed by glucose oxidation and lactate production, was significantly depressed. Pyruvate and octanoate oxidation were unaltered. Oxygen consumption was also unchanged by short-term hypoxia and reoxygenation. With reoxygenation after 30 min of oxygen deprivation, more exaggerated changes in glucose metabolism were noted as well as a depression in pyruvate oxidation and unaltered octanoate oxidation. Oxidation of octanoate was slightly depressed after 60 min of hypoxia. Cell viability assessed after reoxygenation was not significantly altered until 60 min of oxygen deprivation. The results indicate that cytosolic changes occur after short periods of hypoxia followed by reoxygenation, whereas mitochondrial function is more resistant to damage inflicted by hypoxia and reoxygenation.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

Metabolic Responses of the Dopaminergic System during Hypoxia in Newborn Brain

The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced F_iO₂ (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.     

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.