Separable detection of lipophilic- and hydrophilic-phase free radicals from the ESR spectrum of nitroxyl radical in transient MCAO mice

Free radicals are believed to be key factors that promote ischemia reperfusion injury in the brain. This study used the characteristic spectrum of methoxycarbonyl-PROXYL to detect free radical reactions in hydrophilic and lipophilic compartments in a transient middle cerebral artery occlusion (MCAO) mouse model. Methoxycarbonyl-PROXYL, which has a high water/octanol partition coefficient, allows the detection of nitroxyl radical in both compartments simultaneously. Free radicals generation was analysed from the enhanced ESR signal decay rate of methoxycarbonyl-PROXYL. The signal decay rate in the lipidic compartment was significantly enhanced 1 h after reperfusion following MCAO. The enhanced signal decay rate was significantly suppressed by Trolox. The accumulation of lipid peroxidation products increased by 6 h post-reperfusion and was suppressed by methoxycarbonyl-PROXYL or Trolox. These results demonstrate that information pertaining to different sites of free radical generation in vivo can be obtained simultaneously and that lipid-derived radicals are generated in transient MCAO mice.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Oxidative stress and protein oxidation in the brain of water drinking and alcohol drinking rats administered the HIV envelope protein, gp120

Possible roles of oxidative stress and protein oxidation on alcohol-induced augmentation of cerebral neuropathy in gp120 administered alcohol preferring rats drinking either pure water (W rats) or a free-choice ethanol and water (E rats) for 90 days. This study showed that peripherally administered gp120 accumulated into the brain, liver, and RBCs samples from water drinking – gp120 administered rats (Wg rats) and ethanol drinking – gp120 administered rats (Eg rats), although gp120 levels in samples from Eg rats were significantly greater than the levels in samples from Wg rats. The brain samples from ethanol drinking-saline administered (EC) and Wg rats exhibited comparable levels of free radicals that were significantly lower than the levels in Eg rats. Peroxiredoxin-I (PrxI) activity in the brain samples exhibited the following pattern: Wg ≫ ≫ WC ≫ EC > Eg. Total protein-carbonyl and carbonylated hippocampal cholinergic neurostimulating peptide precursor protein levels, but not N -acetylaspartate or N -acetyl aspartylglutamate or total protein-thiol levels, paralleled the free radical levels in the brain of all four groups. This suggests PrxI inhibition may be more sensitive indicator of oxidative stress than measuring free radicals or metabolites. As PrxI oxidation in WC, Wg, and EC rats was reversible, while PrxI oxidation in Eg rats was not, we suggest that alcohol drinking and gp120 together hyperoxidized and inactivated PrxI that suppressed free radical neutralization in the brain of Eg rats. In conclusion, chronic alcohol drinking, by carbonylating and hyperoxidizing free radical neutralization proteins, augmented the gp120-induced oxidative stress that may be associated with an increase in severity of the brain neuropathy.     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage

Recent work suggests that oxygen radicals may be important mediators of damage in a wide variety of pathologic conditions. In this review we consider the evidence supporting the participation of oxygen radicals in the adult respiratory distress syndrome, in ischemia reperfusion injury in the myocardium, and in cerebral vascular injury in acute hypertension and traumatic brain injury. In the adult respiratory distress syndrome there is active sequestration of polymorphonuclear neutrophils in the pulmonary vascular system. There is evidence that activation of these neutrophils results in the production of oxygen radicals which injure the capillary membrane and increase permeability, leading to progressive hypoxia and decreased lung compliance which are hallmarks of the syndrome. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. The metabolism of arachidonic acid is the source of oxygen free radical production in these conditions. In myocardial ischemia and reperfusion injury, the ischemic myocyte is "primed" for free radical production. With reperfusion and reintroduction of molecular oxygen there is a burst of oxygen radical production resulting in extensive tissue destruction. Myocardial ischemia--reperfusion injury shares in common with the other two syndromes activation of the arachidonic acid cascade and acute inflammation. Thus it would appear that the generation of toxic oxygen species may represent a final common pathway of tissue destruction in several pathophysiologic states.     

Free radicals, mitochondria, and hypoxia-ischemia in the developing brain

The immature brain is particularly susceptible to free radical injury because of its poorly developed scavenging systems and high availability of iron for the catalytic formation of free radicals. Neurons are more vulnerable to free radical damage than glial cells, but oligodendrocyte progenitors and immature oligodendrocytes in very prematurely born infants are selectively vulnerable to depletion of antioxidants and free radical attack. Reactive oxygen and nitrogen species play important roles in the initiation of apoptotic mechanisms and in mitochondrial permeability transition, and therefore constitute important targets for therapeutic intervention. Oxidative stress is an early feature after cerebral ischemia and experimental studies targeting the formation of free radicals demonstrate various degrees of protection after perinatal insults. Oxidative stress-regulated release of proapoptotic factors from mitochondria appears to play a much more important role in the immature brain. This review will summarize and compare with the adult brain some of the current knowledge of free radical formation in the developing brain and its roles in the pathophysiology after cerebral hypoxia-ischemia.     

Perturbations in cerebral oxygen radical formation and membrane order following vitamin E deficiency

The effects of dietary vitamin E deficiency on mouse cerebral membrane order and oxygen reactive species were studied. Quantitation of vitamin E levels in several brain regions showed greatest deficiencies in striatum and cerebellum, followed by substantia nigra, and cortex. Vitamin E deficiency increased central-core membrane order in cerebral P2 fraction, but was without effect in the superficial hydrophilic membrane domain. Oxygen radical formation was studied using the probe 2',7'-dichlorofluorescein diacetate. Basal generation rates of oxygen reactive species were 2.5-fold higher when compared to control animals. While hepatic levels of vitamin E are much more reduced than brain levels, in deficient mice, the rate of oxygen radical formation in the liver was unaltered. This implies an special susceptibility of the brain to deficiency of this lipophilic antioxidant vitamin. Data demonstrate that endogenous levels of free radical scavengers, such as vitamin E, may play an important role in maintaining basal oxygen radical levels and membrane integrity. The dietary vitamin E depletion paradigm suggests that a relation exists between elevated levels of oxygen radicals and more rigid hydrophobic central-cores in cerebral membranes, effects that may play a role in mechanisms underlying the neuropathologic lesions observed following vitamin E deficiency.     

Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N ^ω-nitro- l -arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO₂ and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na⁺,K⁺-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

Detection of Free Radicals by Microdialysis/Spin Trapping Epr Following Focal Cerebral Ischemia-Reperfusion and a Cautionary Note on the Stability of 5,5-Dimethyl-1-Pyrroline N-Oxide (DMPO)

We have examined free radical production in a rat model of focal cerebral ischemia using microdialysis coupled with EPR analysis. A microdialysis probe was inserted 2 mm into the cerebral cortex, supplied by the right middle cerebral artery (MCA), and after a 2-hour washout period with artificial cerebral spinal fluid (ACSF), the perfusate solution was changed to ACSF containing the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). No free radicals were detected by DMPO during the pre-ischemia period. Both common carotid arteries and the right MCA were then ligated for 90 minutes. Microdialysate collected every 15 min during the ischemic period demonstrated predominantly superoxide or peroxyl radical production. After release of the occlusive sutures, hydroxyl radical became apparent initially, then thiyl and carbon centered radicals appeared later in samples collected every 15 min for two hours following cortical reperfusion. Careful studies on the purification and stability of DMPO solution were performed to circumvent artifacts and spurious signals.     

Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca⁺⁺-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO₂ = 21%, n = 5) or hypoxic (FiO₂ = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO₄.7H₂O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca²⁺- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Direct measurement of free radicals in the brain cortex and the blood serum after nociceptive stimulation in rats

The concentrations of ROS were measured in samples of the sensorimotor brain cortex and in the rat blood. We measured the following parameters: The six lines spectra, nitroxide radical, free hydroxyl radical and singleton oxygen. Their concentration was measured under physiological conditions, after the nociceptive stimulation and after the application of melatonin, both in normal and stimulated animals. In the brain cortex only the singleton oxygen decreased after the nociceptive stimulation, whereas the nitroxide radicals and six lines spectra increased. The free hydroxyl radicals did not change significantly. In the blood serum the six lines spectra and nitroxide radical increased, the concentration of the free hydroxyl radicals did not change. Melatonin increased both the hydroxyl and nitroxide radicals. There was a non-significant decrease in the six lines spectra. The estimation of ROS can be used as a tool for detecting metabolic changes and the consequences of different environmental influences, in our case the influence of nociception and melatonin.     

Free radical oxidation and antiradical protection of the rat brain during adaptation to intense physical loading

The cerebral free radical oxidation processes on 40 Wistar rats-males were studied by evaluation of thiobarbituric-active products of lipid peroxidation level, superoxide dismutase and glutathione peroxidase activity in sensomotor cortex, hypothalamus and brain stem. Was found that differential stability of rats to motor activities during single intensive physical loading is due by reactivity of free radicals oxidation, associated with decrease of cerebral antioxidant enzymes activity. Long term intensive physical loading may accompanied by reducing of reserve possibilities of antioxidant enzymes an cerebral structures, what possible play potential role in pathogenetic mechanisms of osteoarthritis.     

Time-dependent changes in superoxide dismutase, catalase, xanthine dehydrogenase and oxidase activities in focal cerebral ischaemia

Time-dependent changes in the activities of antioxidant enzymes and an oxidant enzyme, xanthine oxidase (XO), were detected in primary and peri-ischaemic brain regions during permanent occlusion of the middle cerebral artery (MCAO) in rats. There were no changes in superoxide dismutase (SOD) and catalase (CAT) activities after 3 h of MCAO, whereas antioxidant enzyme activities decreased significantly in ischaemic brain areas following 24 h of ischaemia. After 48 h, the enzyme activities returned to the baseline but then a further increase was observed in ischaemic brain areas by 72 h post-ischaemia. Normally, XO exists as a dehydrogenase (XD), but it is converted to XO which contributes to injury in some ischaemic tissues. The XO activity increased slightly at 3 h after ischaemia, but after 24 h of ischaemia it returned to the baseline and then remained relatively unchanged in ischaemic areas. Pretreatment with allopurinol before ischaemia prevented changes in SOD and CAT activities and attenuated brain oedema during 24 h of ischaemia. Neither XO nor XD activity changed in allopurinol-treated rats at the times of ischaemia. These results indicated that ischaemic brain tissue remained vulnerable to free radical damage for as long as 48 h after ischaemia, and XO was probably not an important source of free radicals in cerebral ischaemia.     

Reoxygenation Injury of Human Brain Capillary Endothelial Cells

1. Many studies have demonstrated that endothelial cells from several species can generate oxygen free radicals when subjected to anoxia and reoxygenation. However, due to the heterogeneity of the endothelium within different organs and species, the effects of superoxide dismutase (SOD), catalase, and allopurinol on reoxygenated cultured cells remain quite controversial.2. This review outlines the possible sources of oxygen free radicals within brain endothelial cells.3. We examine the aspects of the effects of SOD catalase and allopurinol on cultured human brain capillary endothelial cells upon reoxygenation.4. Also, we introduce briefly a method of culturing human brain capillary endothelial cells and present our experimental results on the effects of SOD, catalase, and allopurinol in these cultured cells following anoxia and reoxygenation.     

Direct Detection of Ascorbyl Radical in Experimental Brain Injury: Microdialysis and an Electron Spin Resonance Spectroscopic Study

Abstract: To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H₂O₂ and FeCl₂ adjacent to the dialysis probe. When the rats were challenged with cold-induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl₂ dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.     

Hydroxyl radical induced cross-linking between phenylalanine and 2-deoxyribose

Hydroxy radicals induce cross-linking between phenylalanine (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. When phenylalanine and 2-deoxyribose radicals were generated in a 1:1 ratio, the predominant interaction was between Phe and dR radicals while the Phe-Phe and dR-dR cross-links were less abundant. The newly discovered cross-link between 2-deoxyribose and phenylalanine may serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.     

The ascorbate-driven reduction of extracellular ascorbate free radical by the erythrocyte is an electrogenic process

Erythrocytes can reduce extracellular ascorbate free radicals by a plasma membrane redox system using intracellular ascorbate as an electron donor. In order to test whether the redox system has electrogenic properties, we studied the effect of ascorbate free radical reduction on the membrane potential of the cells using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. It was found that the erythrocyte membrane depolarized when ascorbate free radicals were reduced. Also, the activity of the redox system proved to be susceptible to changes in the membrane potential. Hyperpolarized cells could reduce ascorbate free radical at a higher rate than depolarized cells. These results show that the ascorbate-driven reduction of extracellular ascorbate free radicals is an electrogenic process, indicating that vectorial electron transport is involved in the reduction of extracellular ascorbate free radical.     

Production, detection, and adaptive responses to free radicals in exercise

Free radicals (particularly oxygen- and nitrogen-centered radicals), and related reactive oxygen and nitrogen species, are generated in cells and tissues during exercise. Mitochondria (actually, 'leakage' of electrons from ubisemiquinone and other electron transport chain components), xanthine oxidase, and phagocytes such as neutrophils may all contribute to free radical production. In this article we review mechanisms of free radical production during exercise and methods for detecting free radicals and related reactive species, during, or immediately following exercise. The evidence presented strongly suggests that free radicals generated during mild to moderate endurance-type exercise actually form part of the mechanism of exercise adaptation that includes extensive biogenesis of muscle mitochondria, increased muscle blood supply, and altered fuel consumption patterns. We suggest, as originally proposed [1], that (at moderately increased levels) free radicals actually act as intracellular signaling molecules to initiate exercise adaptation. In contrast, endurance exercise of extreme duration and extreme intensity appears to generate much higher levels of free radicals that overwhelm cellular antioxidant defenses, and cause tissue damage. Such free radical damage requires effective protein, lipid, and DNA repair systems, and sufficient recuperation, before exercise adaptation can recommence.