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.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

Noninvasive detection of hydroxyl radical generation in lung by diesel exhaust particles

Diesel exhaust particles (DEP) induce pulmonary tumors, asthma-like symptoms, and the like in experimental animals. The involvement of reactive oxygen species (ROS) is suggested in the injuries induced by DEP, though the generation of ROS has not been proven. The present study provided the first direct evidence of *OH generation in the lungs of living mice after intratracheal instillation of DEP, using noninvasive L-band ESR spectroscopy and a membrane-impermeable nitroxyl probe. *OH generation is confirmed with the enhancement of in vivo ESR signal decay rate of the probe. The decay rate at mid-thorax was significantly enhanced in DEP-treated mice compared to that in vehicle-treated mice. The enhancement was completely suppressed by the administration of either *OH scavengers, catalase, or desferrioxamine, while the administration of SOD further increased the rate. The administration of Fenton's reagents into the lung also enhanced the decay rate of the probe at mid-thorax of mice. These results clearly provided evidence that the intratracheal exposure to DEP in mice produced *OH in the lung through an iron-catalyzed reaction of superoxide/H(2)O(2). This first direct evidence of *OH generation in DEP-treated mice lung may be utilized to determine treatments for DEP-induced lung injury.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

Noninvasive evaluation of in vivo free radical reactions catalyzed by iron using in vivo ESR spectroscopy.

The noninvasive, real time technique of in vivo electron spin resonance (ESR) spectroscopy was used to evaluate free radical reactions catalyzed by iron in living mice. The spectra and signal decay of a nitroxyl probe, carbamoyl-PROXYL, were observed in the upper abdomen of mice. The signal decay was significantly enhanced in mice subcutaneously loaded with ferric citrate (0.2 micromol/g body wt) and the enhancement was suppressed by pre-treatment with either desferrioxamine (DF) or the chain breaking antioxidant Trolox, but only slightly suppressed by the hydroxyl radical scavenger DMSO. To determine the catalytic form of iron, DF was administered at different times with respect to iron loading: before, simultaneously, and after 20 and 50 min. The effect of DF on signal decay, liver iron content, iron excretion, and lipid peroxidation (TBARs) depended on the time of the treatment. There was a good correlation between the signal decay, iron content, and lipid peroxidation, indicating that "chelatable iron" contributed to the enhanced signal decay. The nitroxyl probe also exhibited in vivo antioxidant activity, implying that the process responsible for the signal decay of the nitroxyl probe is involved in free radical oxidative stress reactions catalyzed by iron.     

Noninvasive study of radiation-induced oxidative damage using in vivo electron spin resonance

Nitroxyl radicals injected into a whole body indicate the disappearance of signal intensity of in vivo electron spin resonance (ESR). The signal decay rates of nitroxyl have reported to be influenced by various types of oxidative stress. We examined the effect of X-irradiation on the signal decay rate of nitroxyl in the upper abdomen of mice using in vivo ESR. The signal decay rates increased 1 h after 15 Gy irradiation, and the enhancement was suppressed by preadministration of cysteamine, a radioprotector. These results suggest that the signal decay of nitroxyl in whole mice is enhanced by radiation-induced oxidative damage. The in vivo ESR system probing the signal decay of nitroxyl could provide a noninvasive technique for the study of oxidative stress caused by radiation in a living body.     

Inhibition of TIM‐4 protects against cerebral ischaemia‐reperfusion injury

TIM‐4 plays an important role in ischaemia‐reperfusion injury of liver and kidney; however, the effects of TIM‐4 on cerebral ischaemia‐reperfusion injury (IRI) are unknown. The purpose of the present study was to investigate the potential role of TIM‐4 in experimental brain ischaemia‐reperfusion injury. In this study, cerebral ischaemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1 hour in C57/BL6 mice. The TIM‐4 expression was detected in vivo or vitro by real‐time quantitative polymerase chain reaction, Western blot and flow cytometric analysis. In vivo, the administration of anti‐TIM‐4 antibodies significantly suppressed apoptosis, inhibited inflammatory cells and enhanced anti‐inflammatory responses. In vitro, activated microglia exhibited reduced cellular proliferation and induced IRI injury when co‐cultured with neurons; these effects were inhibited by anti‐TIM‐4 antibody treatment. Similarly, microglia transfected with TIM‐4 siRNA and stimulated by LPS + IFN‐γ alleviated the TIM‐4‐mediated damage to neurons. Collectively, our data indicate that the inhibition of TIM‐4 can improve the inflammatory response and exerts a protective effect in cerebral ischaemia‐reperfusion injury.     

Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke

Studies suggest iron exacerbates the damage caused by ischemic stroke. Our aim was to elucidate the effect of iron overload on infarct size after middle cerebral artery occlusion (MCAO) and to evaluate the efficacy of tempol, a superoxide dismutase mimetic, as a neuroprotective agent. Rats were administered iron +/- tempol before MCAO; control rats received saline. The middle cerebral artery was occluded for 24 h, and the size of the resultant infarct was assessed and expressed as the percentage of the hemisphere infracted (%HI). Iron treatment increased infarct size compared with control (51.83 +/- 3.55 vs. 27.56 +/- 3.28%HI iron treated vs. control, P = 0.01); pretreatment with tempol reversed this (51.83 +/- 3.55 vs. 26.09 +/- 9.57%HI iron treated vs. iron + tempol treated, P = 0.02). We hypothesized that reactive oxygen species (ROS) were responsible for the iron-induced damage. We measured ROS generated by exogenous iron in brain and peripheral vasculature from rats that had not undergone MCAO. There was no increase in ROS production in the brain of iron-treated rats or in brain slices incubated with iron citrate. However, ROS generation in carotid arteries incubated with iron citrate was significantly increased. ROS generation from the brain was assessed after MCAO by dihydroethidine staining; there was a dramatic increase in the ROS generation by the brain in the iron-treated rats compared with control 30 min after MCAO. We propose that iron-induced ROS generation in the cerebral vasculature adds to oxidative stress during an ischemic episode after the disruption of the blood-brain barrier.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.     

In vivo detection of free radicals induced by diethylnitrosamine in rat liver tissue

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used alpha-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration.     

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.     

Determination of reactive oxygen species associated with the degeneration of dopaminergic neurons during dopamine metabolism

Abstract

Oxidative stress is believed to be an important mechanism underlying dopamine-induced neuronal damage. This study provides X-band electron spin resonance (ESR) spectroscopic evidence for reactive oxygen species (ROS) generation during dopamine metabolism. The authors induced excess dopamine metabolism in the mouse striatum by bathing it in tyramine-containing perfusate using microdialysis. The addition of tyramine to the perfusate raised the levels of extracellular dopamine and hydrogen peroxide significantly. The ESR signal from hydroxy-TEMPO decayed during tyramine perfusion and treatment with a monoamine-oxidase inhibitor or radical scavenger suppressed the signal decay. Decreases in the number of tyrosine hydroxylase-immunopositive fibres and in dopamine concentration after tyramine perfusion were observed. Moreover, the tyramine-perfused mice showed a marked methamphetamine-induced rotational response. Notably, these effects of tyramine were suppressed by the simultaneous perfusion of hydroxy-TEMPO. These findings indicate that the ROS generation, which was monitored by hydroxy-TEMPO, caused oxidative damage to the dopaminergic neurons.     

Sesamin attenuates behavioral,biochemical and histological alterations induced by reversible middle cerebral artery occlusion in the rats

Restoration of blood flow to an ischemic brain region is associated with generation of reactive oxygen species (ROS) with consequent reperfusion injury. ROS cause lipid peroxidation, protein oxidation, and DNA damage, all of which are deleterious to cells. So diminishing the production of free radicals and scavenging them may be a successful therapeutic strategy for the protection of brain tissue in cerebral stroke. The present study investigated the neuroprotective effect of sesamin (Sn) to reduce brain injury after middle cerebral artery occlusion (MCAO). The middle cerebral artery (MCA) of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. Sesamin is the most abundant lignan in sesame seed oil is a potent antioxidant. Sesamin (30 mg/kg) was given orally twice, 30 min before the onset of ischemia and 12 h after reperfusion. The initial investigations revealed that sesamin reduced the neurological deficits in terms of behavior and reduced the level of thiobarbituric acid reactive species (TBARS), and protein carbonyl (PC) in the different areas of the brain when compared with the MCAO group. A significantly depleted level of glutathione and its dependent enzymes (glutathione peroxidase [GPx] and glutathione reductase [GR]) in MCAO group were protected significantly in MCAO group treated with sesamin. The present study suggests that sesamin may be able to attenuate the ischemic cell death and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the rat brain. Thus, sesamin may be a potential compound in stroke therapy.     

Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation. Radiation effect on nitroxyl decay of a lipophilic spin probe in the head region

X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood-brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.     

Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation: Radiation effect on nitroxyl decay of a lipophilic spin probe in the head region

X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood–brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.     

Calcitonin gene-related peptide prevents blood-brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion

Cerebral ischemia is one of the diseases that most compromise the human species. Therapeutic recovery of blood-brain barrier (BBB) disruption represents a novel promising approach to reduce brain injury after stroke. To determine the effects of calcitonin gene-related peptide (CGRP) on the BBB participate in stroke progression, rat cerebral ischemia reperfusion injury was induced by a 2-hour left transient middle cerebral artery occlusion (MCAO) using an intraluminal filament, followed by 46h of reperfusion. CGRP (1μg/ml) at the dose of 3μg/kg (i.p.) was administered at the beginning of reperfusion. Subsequently, 48h after MCAO, arterial blood pressure, infarct volume, water content, BBB permeability, BBB ultrastructure, levels of aquaporin-4 (AQP4) and its mRNA were evaluated. CGRP could reduce arterial blood pressure (P<0.001), infarct volume (P<0.05), cerebral edema (P<0.01), BBB permeability (P<0.05), AQP4 mRNA expression (P<0.05) and AQP4 protein expression (P<0.01). Furthermore, CGRP treatment improved ultrastructural damage of capillary endothelium cells and decreased the loss of the tight junction observed by transmission electronic microscopy (TEM) after 46h of reperfusion. Our findings show that CGRP significantly reduced postischemic increase of brain edema with a 2-hour therapeutic window in the transient model of focal cerebral ischemia. Moreover, it seems that at least part of the anti-edematous effects of CGRP is due to decrease of BBB disruption by improving ultrastructural damage of capillary endothelium cells, enhancing basal membrane, and inhibiting AQP4 and its mRNA over-expression. The data of the present study provide a new possible approach for acute stroke therapy by administration of CGRP.     

Transient focal cerebral ischemia/reperfusion induces early and chronic axonal changes in rats: its importance for the risk of Alzheimer's disease

The dementia of Alzheimer's type and brain ischemia are known to increase at comparable rates with age. Recent advances suggest that cerebral ischemia may contribute to the pathogenesis of Alzheimer's disease (AD), however, the neuropathological relationship between these two disorders is largely unclear. It has been demonstrated that axonopathy, mainly manifesting as impairment of axonal transport and swelling of the axon and varicosity, is a prominent feature in AD and may play an important role in the neuropathological mechanisms in AD. In this study, we investigated the early and chronic changes of the axons of neurons in the different brain areas (cortex, hippocampus and striatum) using in vivo tracing technique and grading analysis method in a rat model of transient focal cerebral ischemia/reperfusion (middle cerebral artery occlusion, MCAO). In addition, the relationship between the changes of axons and the expression of β-amyloid 42 (Aβ42) and hyperphosphorylated Tau, which have been considered as the key neuropathological processes of AD, was analyzed by combining tracing technique with immunohistochemistry or western blotting. Subsequently, we found that transient cerebral ischemia/reperfusion produced obvious swelling of the axons and varicosities, from 6 hours after transient cerebral ischemia/reperfusion even up to 4 weeks. We could not observe Aβ plaques or overexpression of Aβ42 in the ischemic brain areas, however, the site-specific hyperphosphorylated Tau could be detected in the ischemic cortex. These results suggest that transient cerebral ischemia/reperfusion induce early and chronic axonal changes, which may be an important mechanism affecting the clinical outcome and possibly contributing to the development of AD after stroke.