Effect of oxygen concentration on the formation of molondialdehyde-like material in a model of tissue ischemia and reoxygenation

This study was conducted to explore the functional relationship between oxygen concentration during tissue reoxygenation after ischemia and the extent of postischemic lipid peroxidation, an indicator of reoxygenation injury. Excised rat liver or kidney tissue was rendered ischemic for 1 h at 37°C, minced into 1 mm³ fragments, and then reoxygenated for 1 h in flasks of buffered salt solution containing various amounts of oxygen. Production of malondialdehyde-like material (MDA) was measured to indicate lipid peroxidation. MDA production was minimal at oxygen tensions less than 10 mmHg, increased sharply from 10 to 50 mmHg, and plateaued at approximately 100 mmHg. A similar functional relationship was produced by a simple mathematical model of free radical mediated lipid peroxidation in biological membranes, suggesting that MDA production is indeed caudes by free radical oxidation of membrane phospholipids and that the oxygen effect is governed by simple competition between chain propagation and chain termination reactions within the membrane. These experimental and analytical results confirm that relatively low concentrations of oxygen are sufficient to produce oxidative damage in post-ischemic tissues.     

Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion

Recent data suggest that oxygen free radicals are implicated in the pathogenesis of ischemic injury. This study evaluates the effects of allopurinol, a xanthine oxidase (XO) inhibitor, on malonaldehyde generation, free sulfhydryl levels, oxygen consumption, and water contents of rat gastrocnemius muscles of female Sprague-Dawley rats subjected to tourniquet shock and after hind-limb reperfusion. Serum lactic dehydrogenase isozyme patterns after ligature release were also examined. Our results show that the four muscle parameters were not altered during 5 hr of ischemia, but that on hind-limb reperfusion, malonaldehyde production, SH levels, O2 consumption, and water contents were significantly altered in the control animals, but not in those pretreated with allopurinol. LDH serum patterns of the untreated animals showed the presence of all five isoforms; these were much less evident in the drug-protected rats. Our data suggest that following ischemia, the affected muscles are unable to recover their normal function when reperfusion is resumed. The subsequent damage is probably due to the generation of cytotoxic superoxide radicals formed during the XO-catalyzed transformation of hypoxanthine to uric acid on tissue reoxygenation. The severity of tissue damage is related to the duration of the ischemic episode possibly due to hypoxanthine accumulation during ischemia.     

Protein S100B release from rat brain slices during and after ischemia: comparison with lactate dehydrogenase leakage

One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca⁺² ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca⁺² ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels.

These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study.     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

The Efficacy of Antioxidants Administered during Low Temperature Storage of Warm Ischemic Kidney Tissue Slices

Accumulation of products of lipid peroxidation (malondialdehyde, conjugated dienes, lipid peroxides, and Schiff bases) was evaluated in rabbit kidney cortex slices made ischemic for 60 min followed by 18 h storage at 5°C in UW Na gluconate solution and 210 min normothermic reoxygenated incubation. In addition, the effect of adding Trolox (1 mM), deferoxamine (1 mM), and ascorbate (1 mM) as supplemental antioxidants to the UW gluconate solution was evaluated. Lipid peroxidation was slightly increased after hypothermic storage compared to slices subjected to ischemia alone but was not significantly different than ischemic slices during subsequent incubation at normothermia. The addition of either deferoxamine or Trolox to the storage solution substantially reduced lipid peroxidation both during hypothermic storage and subsequent to normothermic incubation. Ascorbate had a mild prooxidant effect as a sole additive to the UW gluconate solution but was clearly prooxidant when combined with either deferoxamine or Trolox. These results suggest that supplemental antioxidants added to the UW gluconate solution under conditions analogous to machine perfusion preservation have a potential role in reducing oxidative stress in kidney tissues harvested after warm ischemia and that hypothermia may be a valuable adjunct to resuscitative therapeutic regimens developed for salvage of ischemic kidneys for transplantation.     

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum

Summary. Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.     

Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide

This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) in aqueous systems. Reaction of HO. with DMSO produces methane sulfinic acid as a primary product, which can be detected by a simple colorimetric assay. To evaluate this method for estimating total HO. production, we studied three model systems, including the Fenton reaction, gamma irradiation of water, and ultraviolet photolysis of hydrogen peroxide, for which the theoretical maximum yield of HO. could be calculated and compared to measured DMSO oxidation. The results confirm that 0.05 to 1 M DMSO may be used to capture nearly all of the expected HO. radicals formed. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in aqueous systems pretreated with DMSO.     

Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: a method to quantify hydroxyl radical formation in physiologic systems

A major impediment to the confirmation of free radical mechanisms in pathogenesis is a lack of direct, chemical evidence that oxygen centered free radicals actually arise in living tissues in quantities sufficient to cause serious damage. This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) under physiologic conditions. Reaction of HO. with DMSO produces methane sulfinic acid (MSA) as a primary product, which can be detected by a simple colorimetric assay. To develop a method for estimating total HO. production, we studied two model systems: the superoxide driven Fenton reaction in vitro, using xanthine oxidase as the source of superoxide, and a computer model of Fenton chemistry. Measured MSA production both in vitro and in the computer model was a predictable function of the concentrations of DMSO and competing scavengers of HO., according to the principle of competition kinetics. Both experimental results and model calculations showed that Scatchard analysis may be used to infer total HO. generation, despite the presence of scavengers other than DMSO, such as mannitol. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in biologic systems pretreated with DMSO, and Scatchard analysis of repeated experiments with varying DMSO concentrations can yield an estimate of total HO. generation.     

Effect of ischemia/reperfusion sequence on cytosolic iron status and its release in the coronary effluent in isolated rat hearts

The hypothesis that oxygen-derived free radicals play an important role in myocardial ischemic and reperfusion injury has received a lot of support. In the presence of catalytic amounts of transition metals such as iron, superoxide anions, and hydrogen peroxide can be transformed into a highly reactive hydroxyl radical °OH (Haber-Weiss reaction). In view of this, we have undertaken this study to investigate whether iron is involved in the reperfusion syndrome and therefore could aggravate free radicals injury. Coronary effluent iron concentrations and cardiac cytosolic iron levels were evaluated in rat hearts subjected to an ischemia/reperfusion sequences. In the case of total ischemia, iron concentration in coronary effluents peaked immediately in the first sample collected upon reperfusion. However, in the case of partial ischemia, iron concentration in coronary effluents peaked rather exclusively during ischemia period. Cardiac cytosolic iron level augmented significantly after 30 min of total ischemia and non significantly in the other ischemia protocols compared to perfused control hearts. It also appears that the iron released is not protein-bound, and could therefore have a marked catalytic activity. The results of the present study suggest that in the oxygen paradox, iron plays an important role in inducing alterations during reoxygenation.     

Neuroprotective effects of Ptychopetalum olacoides Bentham (Olacaceae) on oxygen and glucose deprivation induced damage in rat hippocampal slices

Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae) are used in traditional medicine by patients presenting age associated symptoms and those recovering from stroke. The aim of this study is to evaluate the neuroprotective properties of PO ethanol extract (POEE) using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation). Mitochondrial activity, an index of cell viability, was assessed by the MTT assay; in addition, the free radicals content was estimated by the use of dichlorofluorescein diacetate as probe. The OGD ischemic condition significantly impaired cellular viability, and increased free radicals generation. In non-OGD slices, incubation with POEE (0.6 microg/ml) increased (approximately 40%) mitochondrial activity, without affecting free radicals levels. In comparison to OGD controls, slices incubated with POEE (0.6 microg/ml) during and after OGD exposure had significantly increased cellular viability. In addition, at this same concentration, POEE prevented the increase of free radicals content induced by OGD. In view of the fact that respiratory chain inhibition and increased generation of free radicals are major consequences of the ischemic injury, this study suggests that Ptychopetalum olacoides contains useful neuroprotective compounds and, therefore, deserves further scrutiny.     

Increased ultra weak chemiluminescence emission from rat heart at postischemic reoxygenation: protective role of vitamin E

Aim of this study was to confirm an increased free radical generation rate during ischemia-reoxygenation, by ultra-weak chemiluminescence detection at the surface of perfused rat heart. We observed that reoxygenation following 30 min global ischemia, induces an increase of ultraweak chemiluminescence emission in isolated perfused heart only if partial depletion of vitamin E is induced by dietary manipulation. Moreover, in normal diet fed rats, vitamin E is partially consumed during global ischemia, but not during reoxygenation. Since chemiluminescence increases during post-ischemic reperfusion, when vitamin E myocardial content is lowered, the most probable free radicals involved are the hydroperoxyl radical derivatives of lipids. These radicals, indeed, are known both to produce photoemission by disproportion and to react with vitamin E. On the other hand, the nature of the reaction that consumes vitamin E during ischemia is still obscure. Accordingly, the basal level of vitamin E myocardial content seems to be a key factor for protecting the heart against reoxygenation injury and its consumption during ischemia could be a determinant of myocardial sensitivity to oxidative stress during reperfusion.     

Free radicals and the etiology of colon cancer

This hypothesis paper reviews diverse evidence suggesting that intracolonic production of oxygen radicals may play a role in carcinogenesis. The hypothesis began to evolve when the author made the chance discovery that 1/10,000 dilutions of feces generated detectable quantities of highly reactive hydroxyl radicals (HO.). The rate of HO. formation, detected using DMSO as a molecular probe, was quite remarkable, corresponding to that which would be produced by over 10,000 rads of gamma irradiation per day, absorbed in the periphery of the fecal mass adjacent to the mucosa. The relatively high concentrations of iron in feces, together with the ability of bile pigments to act as iron chelators that support Fenton chemistry, may very well permit efficient HO. generation from superoxide and hydrogen peroxide produced by bacterial metabolism. Such free radical generation in feces could provide a missing link in our understanding of the etiology of colon cancer: the oxidation of procarcinogens either by fecal HO., or by secondary peroxyl radicals (ROO.) to form active carcinogens or mitogenic tumor promotors. Intracolonic free radical formation may explain the high incidence of cancer in the colon and rectum, compared to other regions of the GI tract, as well as the observed correlations of a higher incidence of colon cancer with red meat in the diet, which increases stool iron, and with excessive fat in the diet, which may increase the fecal content of procarcinogens and bile pigments.     

Ketamine abolishes ischemic preconditioning through inhibition of K(ATP) channels in rabbit hearts

Although ketamine inhibits ATP-sensitive K (K(ATP)) channels in rat ventricular myocytes and abolishes the cardioprotective effect of ischemic preconditioning in isolated rat hearts and in rabbits in in vivo, no studies to date specifically address the precise mechanism of this prevention of ischemic preconditioning by ketamine. This study investigated the mechanism of the blockade of ischemic preconditioning by ketamine in rabbit ventricular myocytes using patch-clamp techniques and in rabbit heart slices model for simulated ischemia and preconditioning. In cell-attached and inside-out patches, ketamine inhibited sarcolemmal K(ATP) channel activities in a concentration-dependent manner. Ketamine decreased the burst duration and increased the interburst duration without a change in the single-channel conductance. In the heart slice model of preconditioning, heart slices preconditioned with a single 5-min anoxia, pinacidil, or diazoxide, followed by 15-min reoxygenation, were protected against subsequent 30-min anoxia and 1-h reoxygenation, and the cardioprotection was blocked by the concomitant presence of ketamine. These data are consistent with the notion that inhibition of sarcolemmal or mitochondrial K(ATP) channels may contribute, at least in part, to the mechanism of the blockade of ischemic preconditioning by ketamine.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Xanthine Oxidase is Not Responsible for Reoxygenation Injury in Isolated-Perfused Rat Heart

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger el ai. Gastroenterology 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60min of hypoxia and 30 minutes of reoxygenation. The infusion of 250/JM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60min of hypoxia. Protection from 30min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100μM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allupurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.     

Dynamics of intracellular calcium and free radical production during ischemia in pyramidal neurons.

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction. We investigated the contribution of mitochondrial dysfunction to free radical (FR) overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced FR generation was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydroRhod-2, revealed that FR production during and after the anoxic episode correlates with the accumulation of mitochondrial calcium. However, the mitochondrial calcium uptake inhibitor Ru360 did not prevent FR generation during ischemia and attenuated it to some degree during reoxygenation. On the other hand, the mitochondrial permeability transition blocker cyclosporinA (CsA) completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented deterioration of neuronal intrinsic membrane properties. CsA had no effect on the accumulation of intracellular calcium during ischemia-reperfusion. Nicotinamide, a blocker of NAD+ hydrolysis, reproduced the CsA effects on FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases. These observations suggest that a major determinant of ischemic FR generation in pyramidal neurons is the uncoupling of the mitochondrial respiratory chain, which may be associated with the mitochondrial permeability transition.     

Effect of ischemia in vivo and oxygen-glucose deprivation in vitro on NOS pools in the spinal cord: comparative study

1. This study was performed to compare both the Ca(2+)-dependent nitric oxide synthase (NOS) activity and the neuronal nitric oxide synthase immunoreactivity (nNOS-IR) in the rabbit lumbosacral spinal cord after 15 min abdominal aorta occlusion (ischemia in vivo) and oxygen-glucose deprivation of the spinal cord slices for 45 and 60 min (ischemia in vitro). All ischemic periods were followed by 15, 30 and 60 min reoxygenation in vitro. 2. Catalytic nitric oxide synthase activity was determined by the conversion of (L)-[(14)C]arginine to (L)-[(14)C]citrulline. Neuronal nitric oxide synthase immunoreactivity in the spinal cord was detected by incubation of sections with polyclonal sheep-nNOS-primary antibody and biotinylated anti-sheep secondary antibody. 3. Our results show that ischemia in vivo and the oxygen-glucose deprivation of spinal cord slices in vitro result in a time-dependent loss of constitutive NOS activity with a partial restoration of enzyme activity during 15 and 45 min ischemia followed by 30 min of reoxygenation. A significant decrease of enzyme activity was found during 60 min ischemia alone, which persisted up to 1 h of oxygen-glucose restoration. The upregulation of neuronal nitric oxide synthase was observed in the ventral horn motoneurons after all ischemic periods. The remarkable changes in optical density of neuronal nitric oxide synthase immunoreactive motoneurons were observed after 45 and 60 min ischemia in vitro followed by 30 and 60 min reoxygenation. 4. Our results suggest that the oxygen-glucose deprivation followed by reoxygenation in the spinal cord is adequately sensitive to monitor ischemia/reperfusion changes. It seems that 15 min ischemia in vivo and 45 min ischemia in vitro cause reversible changes, while the decline of Ca(2+)-dependent nitric oxide synthase activity after 60 min ischemic insult suggests irreversible alterations.     

Acidic reoxygenation protects against endothelial dysfunction in rat aortic rings submitted to simulated ischemia

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.     

Oxygen tension regulates the maturation of the blood-brain barrier.

The oxygen tension during the development of vascular systems influences vascular vessel formation through regulating angiogenesis. We studied the effect of hypoxia/reoxygenation (H/R) to explain its role in concert with astrocytes involvement in the development of the blood-brain barrier (BBB). On the basis of the fact that the disappearance of hypoxic regions and the decreased expression of vascular endothelial growth factor (VEGF) were observed by immunohistochemistry in a development-dependent manner in rat cerebral cortex, we examined the effects of astrocytes on the BBB-like properties of ECV304 cells by exposing astrocytes to H/R. Conditioned medium of reoxygenated astrocytes inhibited [(3)H]thymidine incorporation and tube formation of ECV 304 cells. When astrocytes were exposed to reoxygenation, the expression of VEGF was reduced, whereas the expression of angiopoietin-1 and thrombospondin-1 was enhanced. Moreover, [(3)H]sucrose permeability assay revealed that astrocytes enhance the barrier function of ECV 304 cells in coculture model within 5 h of reoxygenation. Correspondingly, the occludin expression of ECV 304 cells was slightly increased by the conditioned medium of reoxygenated astrocytes. In conclusion, our study suggests that reoxygenation of astrocytes may act as a significant driving force for the maturation of the BBB during brain development through oxygen-regulated gene(s).