Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.     

Management of combined penetrating cardiac and abdominal trauma

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography-mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl-alpha-phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, .OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of .OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Oxidative stress following traumatic brain injury in rats

BACKGROUND: Free radicals may be involved in the pathophysiology of traumatic brain injury (TBI) through oxidative damage of neurovascular structures. Endogenous antioxidants, such as ascorbate and alpha-tocopherol, may play a critical role in combating these oxidative reactions and their oxidized products can serve as an important index of oxidative stress. METHODS: We used electron spin resonance (ESR) spectroscopy and in vivo spin trapping (reaction of an organic compound with free radical species) to detect the possible generation of free radicals after TBI. Injury was inflicted by a weight drop technique over the head (5.7 kg-cm). Rats were intravenously infused with either 1 mL, 0.1 M of the spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), or an equivalent volume of saline immediately before TBI or sham-injury. Animals were divided into four groups: (1) Group I: PBN-infused sham-injured, (2) Group II: PBN-infused injured, (3) Group III: saline-infused sham-injured, and (4) Group IV: saline-infused injured. Additional groups of saline-infused uninjured, saline-infused, and PBN-infused injured animals were used for histopathology. Sixty minutes after TBI or sham-injury, rats were again anesthetized and decapitated. The brains were removed within 1 minute, homogenized, and extracted for lipids. The extracts were analyzed by ESR spectroscopy. Brain ascorbic acid (AA) concentration was determined spectrophotometrically, using the ascorbate oxidase assay. RESULTS: No PBN spin adduct signals (indicating trapped free radical species) were visible 60 minutes after TBI. All groups of rats showed an ascorbyl free radical signal. The ascorbyl signal intensity (AI) was, however, significantly higher in the injured rats, while the brain (AA) was significantly reduced. In addition, the ratio of AI/AA, which eliminates the effect of variable ascorbate concentrations in the brain, was also significantly higher in the injured animals. CONCLUSIONS: We conclude that 60 minutes following TBI there was a significantly increased level of oxidative stress in the brain. This may reflect formation of free radical species with subsequent interaction with ascorbate (antioxidant) during the 60 minute period. The lack of PBN spin adduct signals 1 hour after TBI may indicate that free radical generation is time dependent and might be detectable earlier or later than the 60 minute period.     

Scavenging effects of dopamine agonists on nitric oxide radicals

It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson's disease (PD), and the level of nitric oxide radical (.NO), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for .NO in an in vitro .NO-generating system using 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propa namine as an .NO donor and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of.NO generated. .NO appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be approximately 23 and 200 microM, respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6-hydroxydopamine-injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges .NO radicals and hence protects against .NO-related cytotoxicity. This ESR spectrometry method using carboxy-PTIO may be useful for screening other drugs that can quench .NO.     

Oxygen free radical generation during in-utero hypoxia in the fetal guinea pig brain: the effects of maturity and of magnesium sulfate administration

Previous studies have shown, employing direct measurements with electron spin resonance (ESR) spectroscopy, that hypoxia induces an increased production of oxygen free radicals (OFR) in the brain of the guinea pig fetus. The present study using the same approach, investigated the effects of maturity and Mg2+-pretreatment on hypoxia-induced OFR formation in the guinea pig fetal brain. The normoxic and the hypoxic groups were exposed for 60 min to 21% or 7% oxygen, respectively. The control group consisted of term fetuses exposed to normoxia (n=7) and hypoxia (n=7). The experimental groups consisted of the following: (a) for the investigation on maturity effect, preterm fetuses (40 days) exposed to normoxia (n=6) or hypoxia (n=6); and (b) for the Mg2+-pretreatment investigation, term fetuses (60 days) exposed to normoxia (n=6) or hypoxia (n=6) following maternal pretreatment with Mg2+ which consisted of an initial bolus of MgSO4 (600 mg/kg, i.p.) 1 h prior to hypoxia followed by a second dose (300 mg/kg, i.p.). Oxygen free radicals were measured by ESR spectroscopy in the fetal cerebral cortical tissue utilizing phenyl-N-tert-butylnitrone (PBN) spin trapping. Fetal brain tissue hypoxia was documented biochemically by decreased tissue levels of ATP and phosphocreatine. In the control group of term fetuses, the cortical tissue from hypoxic fetuses showed a significant increase in spin adducts (71% increase, p<0.01). In the preterm group, the cortical tissue from hypoxic fetuses showed a 33% increase in spin adducts (p<0.001). The baseline free radical generation during normoxia was 22.5% higher at preterm than at term (41.4+/-3.5 units/g issue vs. 33.8+/-9.3 units/g tissue, p<0.05). In Mg2+-treated groups, spin adduct levels in cortical tissue from hypoxic fetuses did not significantly differ from those of the normoxic group (30.2+/-9.9 units/g tissue, normoxic-Mg2+ vs. 30. 6+/-8.1 units/g tissue, hypoxic-Mg2+). The results indicate that the fetal brain at term may be more susceptible to hypoxia-induced free radical damage than at preterm and that Mg2+ administration significantly decreased the hypoxia-induced increase in oxygen free radical generation in the term fetal guinea pig brain in comparison with non-treated hypoxic group.     

GA strategy for variable selection in QSAR studies: GA-based PLS analysis of calcium channel antagonists

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.     

Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of PAR and 3,5-diCH3-, 3,5-diC2H5-, 3,5-ditC4H9-, 3,5-diOCH3-, 3,5-diSCH3-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H2O2). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicals were detected in rat liver cytochrome P450-containing microsomal or reconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH3-, 3,5-diOCH3-, 3,5-diF- and 3,5-diCl-PAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atom abstraction from the phenolic hydroxyl group is favoured by approximately 125 kJ/mol over an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (+/-85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase/H2O2 incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5-disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.     

Inhibition of oxidation of low density lipoprotein by troglitazone

The effect of a new oral hypoglycemic agent troglitazone, (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)benz yl]-2,4-thiazolidinedione as an antioxidant against the free radical-mediated oxidation of low density lipoprotein (LDL) was studied. The oxidation of LDL gives cholesteryl ester hydroperoxide and phosphatidylcholine hydroperoxide as major primary products. Troglitazone incorporated exogenously into LDL inhibited the oxidations of LDL induced by either aqueous or lipophilic peroxyl radicals and suppressed the formation of lipid hydroperoxides efficiently. Ascorbic acid added into the aqueous phase spared both endogenous alpha-tocopherol and troglitazone in LDL. It was also found by absorption spectroscopic and electron spin resonance (ESR) studies that troglitazone reacted rapidly with a galvinoxyl radical to give a chromanoxyl radical which gives the same ESR spectrum as alpha-tocopherol. This ESR spectrum disappeared rapidly when ascorbic acid was added into the system. These results show that troglitazone acts as a potent antioxidant and protects LDL from oxidative modification.     

Hypoglycemia, hypoxia, and ischemia in a corticostriatal slice preparation: electrophysiologic changes and ascorbyl radical formation

Experimental and clinical data suggest that oxygen and/or glucose deprivation alters electrical transmission in the brain and generates free radicals, which may mediate neuronal death. We have analyzed the effects of oxygen and/or glucose deprivation on both excitatory transmission, by measuring field potential amplitude, and free radical production, by using electron spin resonance (ESR) spectroscopy, in a corticostriatal slice preparation. Combined oxygen and/or glucose deprivation (ischemia) lasting 10 to 20 minutes induced a long-term depression of field potential amplitude. The ascorbyl radical could only be detected in brain slices during the reperfusion-phase after 30 minutes of ischemia. It appeared in the early minutes after the washout of ischemic medium and remained stable throughout the reperfusion phase. This radical was never detected in the external medium. Ischemia induced only a slight, but progressive, release of lactate dehydrogenase (LDH) into the external medium during the reperfusion phase. In contrast, exposure of slices to hypoxia or hypoglycemia alone resulted in transient depression of field potential amplitude, and no generation of ascorbyl radicals was observed on reperfusion. We propose that the long-lasting loss of electrical signals is the early sign of neuronal damage during ischemia. On the other hand, ascorbyl radical formation may be considered an indicator of neuronal injury after prolonged energy deprivation.     

In vivo analysis of hydrogen peroxide and lipid radicals in the striatum of rats under long-term administration of a neuroleptic

It has been hypothesized that free radicals play a causative role in tardive dyskinesia, which is an inveterate movement disorder caused by chronic administration of neuroleptics. To verify this hypothesis, rats were reared while being regularly treated with water containing a neuroleptic, haloperidol (HPD), for 1 year (HPD group). The changes in the striatal hydrogen peroxide content of the rats in the HPD and control groups were measured by using a Pt-disk microelectrode while the animals were in a freely moving state following intraperitoneal administration of HPD (HPD challenge). We also performed electron spin resonance (ESR) detection of lipid radicals in the striatum before the HPD challenge. HPD challenge led to significant elevation of the intrastriatal hydrogen peroxide in all animals, but the elevation in the HPD group was smaller than that in the control group. However, in the HPD group, marked ESR signals of intrastriatal lipid radicals were observed. We think that these results support the hypothesis on the role of free radicals in tardive dyskinesia.     

Cocaine does not compromise cerebral or myocardial oxygen delivery in fetal sheep

Aprikalim, a K+ ATP channel opener, is a potent vasodilator with demonstrated cardioprotective properties against ischemia/reperfusion injury. It is still unknown if K+ ATP channel openers exert their beneficial effects via interaction with oxygen-derived free radicals. Therefore, we investigated the cardioprotective effects of aprikalim against oxygen-derived free radicals. Isolated rabbit hearts were perfused at constant pressure (85 cm H2O) or constant flow (30-35 ml/min). Heart rate, left ventricular developed pressure (LVDP), and either coronary flow or coronary perfusion pressure (CPP) were monitored. Free radicals were produced by electrolysis of the perfusate (0.6 mA, direct current), and 10 microM aprikalim was infused before and after exposure to free radicals. In the constant perfusion pressure experiments, 10 min of exposure to free radicals resulted in a significant reduction of heart rate (137 to 129 beats/min), LVDP (112 to 91 mmHg) and coronary flow (37 to 29 ml/min); coronary flow was more markedly impaired than contractile function. Acetylcholine-induced coronary dilation was also significantly attenuated in the presence of free radicals. After 30 min of recovery, both coronary flow and LVDP were still significantly decreased while acetylcholine-induced coronary dilation had fully recuperated. Aprikalim completely abated the coronary and cardiac depressant actions of free radicals. Constant flow experiments indicated that exposure to free radicals increased CPP (+40%, p < 0.05), an effect totally suppressed by aprikalim. These results demonstrate that aprikalim reverses the cardiodepressant actions of free radicals. The cardioprotection it afforded involves both contractile function and the coronary vasculature. Acetylcholine-induced coronary dilation was blunted by free radicals, an indication of complex interactions at the coronary endothelial level.     

Interaction of UV light-induced alpha-tocopherol radicals with lipids detected by an electron spin resonance prooxidation effect

The reaction rate constants of the interaction between light-induced alpha-tocopherol radicals with unsaturated lipids in a heterogeneous system compared to a homogeneous system are of the same order of magnitude. The decay rates of compartmentalized alpha-tocopherol radicals were significantly reduced by using negatively charged sodium dodecyl sulfate (SDS) micelles. A partially resolved electron spin resonance (ESR) hyperfine structure was observed under the conditions of both high lipid concentrations in comparison to the alpha-tocopherol concentration and of a regular distribution of alpha-tocopherol molecules inside the heterogeneous lipid structures. Alpha-tocopherol radicals have a considerable prooxidation potential at higher concentrations. Ascorbic acid dissolved in the aqueous medium provokes very fast alpha-tocopherol radical recycling through the boundary layer between the aqueous medium and micelles. By contrast, very slow reactions such as those of alpha-tocopherol radicals with glutathione through this boundary layer are measurable. Despite using the heterogeneous SDS micellar system, the decay kinetics of the alpha-tocopherol radical ESR signal is simply compounded. In addition to the known stabilization effect of cholesterol in membrane systems, cholesterol itself acts as a target molecule attacked by free radicals, e.g. alpha-tocopherol radicals. Using stratum corneum extracts that contain unsaturated lipids and cholesterol the alpha-tocopherol radical can prooxidatively react with these compounds. Using focused UV light generates a high radical yield in a relatively short time compared to the lifetime of the alpha-tocopherol radicals. The decay processes after radical induction can be characterized as consecutive reactions. The compartmentalization of radicals induced in SDS micelles and the close proximity of target molecules are essential if very slow one-electron reductions are to be measured.     

The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR)--spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.     

Temporal brain imaging by a rapid scan ESR-CT system in rats receiving intraperitoneal injection of a methyl ester nitroxide radical

We performed in vivo ESR-CT (electron spin resonance-computed tomography) on rats' heads, in which the blood-brain barrier-permeable nitroxide radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) was injected intraperitoneally, using a rapid scan ESR-CT system operating at 700 MHz. In a spatiotemporal study we found that different regions of the brain showed differences in the rate of decay of the radical. Repeated injection of PCAM gave clear ESR-CT images of the brain. We think that the present method is useful for evaluating the capacity to eliminate exogenous free radicals in some parts of the brain.     

Nitrone spin-traps block calcium channels and induce pulmonary artery relaxation independent of free radicals

Free radicals react with nitrones to form stable nitroxides which can be identified by ESR spectroscopy. Unfortunately, little is known regarding the pharmacological properties of these compounds. In this study, three commonly used nitrones, 5,5-dimethylpyrroline-N-oxide (DMPO), alpha-phenyl-tert-butylnitrone (PBN), and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), were found to induce relaxation of preconstricted isolated rat pulmonary artery rings. Additional experiments with PBN indicated that vasorelaxation could not be attributed to production of endothelial derived factors, prostaglandins, or free radicals. Patch-clamp techniques revealed reversible calcium channel blockade with PBN at a concentration below that needed to detect free radicals. Calcium channel blockade probably accounts for the vasorelaxation observed in the isolated ring preparations described here, and should be considered when using nitrone spin-traps both in in vivo and clinical studies.     

Effect of oxidative stress on brain damage detected by MRI and in vivo 31P-NMR

The brain is susceptible to oxidative stress. This is due to the high content of polyunsaturated fatty acids, high rate of oxygen consumption, regional high concentrations of iron, and relatively low antioxidant capacity. These factors may predispose the premature infant to brain damage. Brain damage may be due to: 1. Brief anoxia followed by hyperoxia (mimics parturition oxidative stress); or 2. Prolonged exposure to hyperoxia (mimics oxidative stress from postpartum maintenance in a hyperoxic environment). We have developed two animal models to examine these forms of oxidative stress on the brains of rats. In Model I rats were exposed to brief anoxic anoxia (100% N2) followed by hyperoxia (100% O2). Using T2-weighted Magnetic Resonance Imaging (MRI) brain intensity decreased following the treatment suggesting water loss or free radical production. In vivo 1H-NMR showed brain water content appeared to increase, however variability rendered this result insignificant. Electron spin resonance (ESR) spin trapping, using a-phenyl-N-tert-butylnitrone (PBN) produced a free radical signal from the anoxic-anoxia hyperoxia treated animals which suggests the decrease in MRI T2-weighted image signal intensity was due to free radicals. In Model II, we examined the effects of prolonged normobaric hyperoxia (85% O2) on blood-brain barrier (BBB) integrity and brain phosphorous metabolism. BBB permeability increased following 1 week of hyperoxia. In addition, measurement of high energy phosphates, using in vivo 31P-NMR, showed the PCr/ATP ratio significantly decreased, the ATP/Pi ratio increased and the (ATP+PCr)/Pi ratio increased. Because the BBB is sensitive to oxidative stress its loss of integrity may be due to free radicals. The level of oxidative stress may result in brain elevation of ATP as an adaptation mechanism. In conclusion, anoxic-anoxia and prolonged hyperoxia exposure produce MRI visible changes in the brain. These two mechanisms may be important in the etiology of brain damage observed in many premature infants.     

Immunogenicity, safety and efficacy of tetravalent rhesus-human, reassortant rotavirus vaccine in Belém, Brazil

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to beta-subunit of hemoglobin (beta-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginine. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (< 1 mmol/L) could protect the heart form the ischemia-reperfusion injury but at higher concentration aggravate the injury. Addition of NAME to the reperfusion solution could also protect the myocardium. Addition of xanthine (X)/xanthine oxidase (XO) or Fe2+/H2O2 to the reperfusion solution increased the production of NO and oxygen free radicals and the ischemia-reperfused injury simultaneously. Addition of superoxide dismutase (SOD) and catalase decreased the production of NO and oxygen free radicals and the ischemia-reperfusion injury.     

Detection of free radicals in blood by electron spin resonance in a model of respiratory failure in the rat

Previous work has suggested that a free radical mechanism is involved in some types of muscle fatigue and that there can be free radicals released extracellularly. Because muscle fatigue may be an important factor in respiratory failure, the authors tested the hypothesis that increased concentrations of free radicals could be detected in the blood of animals undergoing severe resistive loading to respiratory failure. An ex vivo spin trapping technique with alpha-phenyl-N-tert-butylnitrone (PBN) was used to investigate the possible formation of free radicals in systemic blood samples by electron spin resonance (ESR) spectrometry. After 2.5-3 h of severe inspiratory resistive loading with 70% supplemental inspired oxygen, free radical levels in the form of PBN-adducts were found to rise significantly over the control group breathing room air and the control group breathing 70% oxygen (p < 0.05, N = 8). There were no significant differences between control groups breathing room air and control groups breathing 70% oxygen. This study presents direct evidence that free radicals are produced ex vivo and that they can be detected in the systemic circulation due to excessive resistive loading of the respiratory muscles.     

Influence of sonographic contrast media on microcirculation in rats

In an open placebo-controlled study the influence of the injection of different sonographic contrast media on the microcirculation was proved. The study was performed in 7 Sprague-Dawley rats. In order to examine this query two different sonographic contrast media in comparison to agitated electrolyte solution (as the placebo) were injected into the abdominal aorta of 7 anaesthetized rats as a 2 ml/kg bolus at 10-min intervals. Examined were a newly developed agitated ultrasound solution (AK I: an aequeous solution of a vegetable phospholipid) and a radiographic contrast agent (AK II: 741 mg Ioversol or 350 mg iodine, respectively, per ml) which is used in an agitated form as ultrasound contrast agent, too. 1 min before and until 2 min after each injection the capillary perfusion of the same vessel area in the major omentum was measured (video-recording by use of intravital microscopy). The erythrocyte velocity was determined off-line by an image analysing system. Whereas the agitated X-ray contrast medium AK II decreases the mean capillary perfusion (temporary flow stagnation in single capillaries), AK I as well as agitated electrolyte solution did not influence the capillary erythrocyte velocity in the major omentum. The gaps which appeared immediately after the injection of AK I seem to have been brought about by spherosomes of AK I. Still the spherosomes are so small that they can pass through the capillaries, or if they are larger which cannot be determined using in vivo microscopy the flow force necessary for the deformation of the bubbles is so small that the capillary perfusion is not influenced. The injection of agitated AK I does not lead to significant changes of the microcirculation.     

Reflex sympathetic dystrophy associated with extraforaminal disc herniation at the L5-S1 level

Physical exercise is known to induce oxidative stress leading to the generation of free radicals. This increased generation of free radicals might lead to lipid peroxidation and tissue damage, more so under deficient/impaired antioxidant states. In the present study, we report the role of vitamin E and selenium (Se) during exercise-induced oxidative stress in the pulmonary tissue. Vitamin E and/or Se deficiency in female albino rats resulted in generation of free radicals as revealed by electron spin resonance (ESR) spectra in the lung tissue, indicating the onset of oxidative stress. When these animals were subjected to a single bout of exhaustive exercise, there was an additional increase in the generation of oxy-free radicals, which might lead to tissue damage. However, no such signals were recorded in the lung tissue of vitamin E- and Se-supplemented animals, when subjected to a similar exercise program, suggesting that protection is offered by vitamin E and Se in combating oxidative stress.