Detection of Free Radical Activity During Transient Global Ischemia and Recirculation: Effects of Intraischemic Brain Temperature Modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two-vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3- and 2,5-DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3- and 2,5-DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3- and 2,5-DHBA levels increased by 2.5- and 2.8-fold, respectively. Levels of 2,3-DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5-DHBA levels persisted for 2 h. The increases in 2,3- and 2,5-DHBA levels observed following hyperthermic ischemia were significantly higher (3.8- and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical-induced injury in the development of ischemic damage.     

Monoamine oxidase-induced hydroxyl radical production and cardiomyocyte injury during myocardial ischemia–reperfusion in rats

To elucidate the involvement of monoamine oxidase (MAO) in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion, we applied microdialysis technique to the heart of anesthetized rats. Dialysate samples were collected during 30?min of induced ischemia followed by 60?min of reperfusion. We monitored dialysate 3,4-dihydrobenzoic acid (3,4-DHBA) concentration as an index of hydroxyl radical production using a trapping agent (4-hydroxybenzoic acid), and dialysate myoglobin concentration as an index of cardiomyocyte injury in the ischemic region. The effect of local administration of a MAO inhibitor, pargyline, was investigated. Dialysate 3,4-DHBA concentration increased from 1.9?±?0.5?nM at baseline to 3.5?±?0.7?nM at 20–30?min of occlusion. After reperfusion, dialysate 3,4-DHBA concentration further increased reaching a maximum (4.5?±?0.3?nM) at 20–30?min after reperfusion, and stabilized thereafter. Pargyline suppressed the averaged increase in dialysate 3,4-DHBA concentration by ～72% during occlusion and by ～67% during reperfusion. Dialysate myoglobin concentration increased from 235?±?60?ng/ml at baseline to 1309?±?298?ng/ml at 20–30?min after occlusion. After reperfusion, dialysate myoglobin concentration further increased reaching a peak (5833?±?1017?ng/ml) at 10–20?min after reperfusion, and then declined. Pargyline reduced the averaged dialysate myoglobin concentration by ～56% during occlusion and by ～41% during reperfusion. MAO plays a significant role in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion.     

Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate-trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham-trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3-DHBA (3.3-fold, p < 0.01), 2,5-DHBA (2.5-fold, p < 0.01), and glutamate (2.8-fold, p < 0.01) compared with controls. The levels of 2,3-DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5-DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3-DHBA and glutamate concentrations ( p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3- and 2,5-DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.     

Role of Reactive Oxygen Species in the Sensitivity of Rat Hypertrophied Myocardium to Ischemia

The relationship between hydroxyl radical (OH*) generation in the zone of ischemia/reperfusion and the size of infarction formed was investigated in 18-22-week-old anaesthetized male SHRSP and Wistar rats using a myocardial microdialysis technique. The marker of OH* generation, 2,3-dihydroxybenzoic acid (2,3-DHBA), was analyzed in dialyzates by high performance liquid chromatography with electrochemical detection. Myocardial ischemia was induced by ligation of the descending branch of the left main coronary artery for 30 min. The mean value of basal 2,3-DHBA level in the dialyzate samples from SHRSP (243 +/- 21 pg for 30 min) was significantly higher than that from Wistar rats (91 +/- 4 pg for 30 min, p < 0.0002); it positively correlated with left ventricular hypertrophy (r = 0.806; p < 0.05). During reperfusion total 2,3-DHBA output was 1.8-fold higher in SHRSP than in Wistar rats (659 +/- 60 pg versus 364 +/- 66 pg for 60 min, respectively, p < 0.0002). At the same time, 2,3-DHBA increase above the basal level was the same in Wistar and SHRSP rats (181 +/- 25 and 172 +/- 36 pg for 60 min, respectively). The infarct size in SHRSP (45.4 +/- 4.3%) was significantly higher (p < 0.05) than in Wistar rats (32.8 +/- 3.3%). There was a significant positive correlation between basal level of 2,3-DHBA and total reperfusion 2,3-DHBA content in SHRSP (r = 0.752; p < 0.05). Thus, data obtained clearly indicate that the hypertrophied myocardium of SHRSP was less tolerant to ischemia/reperfusion than that of Wistar rats due to chronically increased OH* production and enhanced total OH* output during reperfusion. Greater myocardial damage in SHRSP than in Wistar rats following the equal increase in OH* production above the basal level suggests the existence of deficit of the antioxidant defense in the hypertrophied myocardium.     

Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion

This study sought to determine whether gallium-desferrioxamine (Ga/DFO) can curb free radical formation and mitigate biochemical and electrophysiological parameters of injury in the cat retina subjected to ischemia followed by reperfusion.For the biochemical studies, cat eyes were subjected to 90 min of retinal ischemia followed by 5 min of reperfusion, and enucleation of one eye of each cat was used to measure retinal reperfusion injury. Before enucleation of fellow eyes, 2.5 mg/kg Ga/DFO was injected intravenously 5 min before reperfusion. The flux of hydroxyl radicals, as measured directly by conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA), was significantly lower in Ga/DFO-treated eyes. The mean normalized level of 2,3-DHBA (considered a specific marker of hydroxyl radicals) was 3.5 times higher in untreated eyes. Ga/DFO caused a significant reduction, by 2.56-fold, in lipid peroxidation, as reflected by levels of malondialdehyde. Ascorbic acid, a natural antioxidant present in the retina, is severely depleted in untreated eyes. In contrast, in Ga/DFO-treated eyes, levels were 10 times higher than the control. Energy charge was 2.38 times higher in treated eyes. Levels of purine catabolites (hypoxanthine, xanthine, and uric acid) that reflect excessive metabolism of purine nucleotides were approximately twice higher in untreated retinas. Electroretionographic studies, performed on a different subset of animals, substantiated the biochemical results. In Ga/DFO-treated eyes the amplitude of the mixed cone-rod response b-wave (as compared with fellow nonischemic eyes) fully recovered within 24 h after ischemia (b-wave ratio 1.04 +/- 0.09, [mean +/- SEM]) whereas ischemic/reperfused and nontreated eyes recovered to only 0.33 +/- 0. 05. The results show that severe biochemical and functional retinal injury occurs in cat eyes subjected to ischemia and reperfusion. These severe changes were significantly reduced by a single administration of Ga/DFO just before reperfusion. We hypothesize that the protection afforded by Ga/DFO is due to a combined effect of "Push-Pull" mechanisms interfering with transition metal-dependent and free radical-mediated injurious processes.     

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.     

Ischemic preconditioning decreases the reperfusion-related formation of hydroxyl radicals in a rabbit model of regional myocardial ischemia and reperfusion: the role of K(ATP) channels

The objective of this study was to assess the effects of ischemic preconditioning (IP) on hydroxyl free radical production in an in vivo rabbit model of regional ischemia and reperfusion. Another goal was to determine whether K_ATP channels are involved in these effects.

The hearts of anesthetized and mechanically ventilated New Zealand White rabbits were exposed through a left thoracotomy. After IV salicylate (100 mg/kg) administration, all animals underwent a 30-min stabilization period followed by 40 min of regional ischemia and 2 h of reperfusion. In the IP group, IP was elicited by 5 min of ischemia followed by 10 min of reperfusion (prior to the 40-min ischemia period). Glibenclamide, a K_ATP channel blocker, was administered prior to the preconditioning stimulus. Infarct size was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We quantified the hydroxyl-mediated conversion of salicylate to its 2,3 and 2,5-dihydroxybenzoate derivatives during reperfusion by high performance liquid chromatography coupled with electro-chemical detection.

IP was evidenced by reduced infarct size compared to control animals: 22% vs. 58%, respectively. Glibenclamide inhibited this cardioprotective effect and infarct size was 53%. IP limited the increase in 2,3 and 2,5-dihydroxybenzoic acid to 24.3 and 23.8% above baseline, respectively. Glibenclamide abrogated this effect and the increase in 2,3 and 2,5-dihydroxybenzoic acid was 94.3 and 85% above baseline levels, respectively, similar to the increase in the control group. We demonstrated that IP decreased the formation of hydroxyl radicals during reperfusion. The fact that glibenclamide inhibited this effect, indicates that K_ATP channels play a key role in this cardioprotective effect of IP.     

On the application of 4-hydroxybenzoic acid as a trapping agent to study hydroxyl radical generation during cerebral ischemia and reperfusion

Aromatic hydroxylation from the reaction between hydroxyl radical and salicylate or its related compounds has been often utilized as a marker for the generation of hydroxyl radicals. We have investigated several technical aspects of applying this method to study hydroxyl radical production during cerebral ischemia and reperfusion using the hydroxylation of 4-hydroxybenzoic acid (4-HBA) to form 3,4-dihydroxybenzoic acid (3,4-DHBA). 4-HBA was administered to rats either through intravenous infusion, or by way of an in vivo microdialysis probe implanted in the brain. Dialysate containing 3,4-DHBA was collected and analyzed by HPLC with electrochemical detection. An endogenous compound was found to co-elute with 3,4 -DHBA but could be separated by varying the chromatographic conditions. Because of interrupted blood flow during cerebral ischemia and reperfusion, delivery of 4-HBA through the microdialysis probe is a preferred method to systemic administration such as intravenous infusion. It is concluded that the oxidation of 4-HBA to 3,4-DHBA can be a reliable and accurate indicator for the formation of hydroxyl radical in vivo if the experiments are well designed to avoid potential pitfalls associated with technical difficulties of the method.     

Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitant with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.     

Effects of α-Phenyl-tert-Butylnitrone and Selegiline on Hydroxyl Free Radicals in Rat Striatum Produced by Local Application of Glutamate

Abstract: The hydroxyl radical is a very reactive oxygen species that damages biomolecules in the brain and in other tissues. The possible pharmacological intervention to prevent hydroxyl radical formation was studied in vivo using the microdialysis technique in brains of nonanesthetized rats. Hydroxyl radicals form stable adducts [mainly 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA)] via an aromatic hydroxylation reaction with salicylic acid. 2,3-DHBA was separated and quantified by HPLC and electrochemical detection. Microdialysis probes were implanted into the striatum 1 day before measurement of levels of hydroxyl radicals. The next day, the probes were first perfused for 120 min with a modified Ringer's solution containing 5 m M salicylic acid, to obtain stable baselines. Afterward, the perfusion solution was switched to another solution that in addition contained 50 m M glutamate, to stimulate radical formation. Twenty minutes later, α-phenyl- tert -butylnitrone (PBN; 100 mg/kg), selegiline (10 mg/kg), or saline was administered intraperitoneally. The glutamate perfusion produced marked two- to 2.5-fold increases in 2,3-DHBA content. Treatment with PBN significantly antagonized the rise of 2,3-DHBA level, indicating that PBN is a direct radical scavenger not only in vitro but also in vivo. Acute treatment with selegiline failed to reduce significantly the glutamate-induced radical formation. The acute experiments presented here do not support the suggestion that the neuroprotective effects of selegiline described in the literature are due to a potential hydroxyl radical scavenging property of the drug.     

Systemic administration of d-amphetamine induces long-lasting oxidative stress in the rat striatum

The long-term effect of d-amphetamine (AMPH) on the induction of oxidative stress was examined in vivo in the rat brain. In this study, 2,3-dihydroxybenzoic acid (2,3-DHBA) and malonaldehyde (MDA) were used as the index of the hydroxyl radical and lipid peroxidation, respectively. The levels of 2,3-DHBA, MDA and dopamine (DA) in striatal homogenates were examined 7 days following injection of a single large dose of AMPH (7.5 mg/kg, i.p.) in rats pretreated with desipramine (10 mg/kg, i.p.), an agent that inhibits the metabolism of AMPH. Our results showed that 2,3-DHBA and MDA levels were significantly increased by AMPH, whereas DA and its metabolites, DOPAC and HVA were depleted in the striatum. Pretreatment with the glutamate NMDA receptor subtype antagonist MK-801 (1 mg/kg, i.p.) attenuated the increases of 2,3-DHBA and MDA, and provided partial protection against the long-lasting loss of DA produced by AMPH. Overall, the results demonstrate that AMPH could induce sustained production of free radical and oxidative damage, and lead to DA terminal degeneration in the striatum of the rat.     

LC/MS analysis of hydroxylation products of salicylate as an indicator of in vivo oxidative stress

Hydroxyl radical attack upon salicylate leads to the generation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and therefore can be used to assess hydroxyl radical formation both in vitro and in vivo. Evidence is presented for a highly sensitive LC/MS assay for the quantification of 2,3-DHBA. Calibration curves showed linearity within the concentration range tested (0.5-6.5 pmol/microl rat plasma) with a coefficient of determination (r2) greater than 0.99. A detection limit of less than 0.25 pmol for 2,3-DHBA has been achieved. The intra-assay and inter-assay variability were determined to be 4.1% and 12.5%, respectively. This method was evaluated for the determination of drug-induced in vivo generation of oxidative stress by means of 1,1,1-trichloroethane (TCE) a compound that is a pseudosubstrate for cytochrome P450 and is known to induce oxygen reductase activity of this enzyme(s). TCE treated rats had a 6.4-fold increase in the mean maximal plasma 2,3-DHBA concentration as compared to the saline treated rats (p = .009). The developed LC/MS assay requires minimal sample preparation and provides a rapid and sensitive method for quantification of 2,3-DHBA as a specific indicator of hydroxyl radical generation.     

Brain Hydroxyl Radical Generation in Acute Experimental Head Injury

Abstract: The time course and intensity of brain hydroxyl radical (^?OH) generation were examined in male CF-1 mice during the first hour after moderate or severe concussive head injury. Hydroxyl radical production was measured using the salicylate trapping method in which the production of 2,3- and/or 2,5-dihydroxybenzoic acid (DHBA) in brain 15 min after salicylate administration was used as an index of ^?OH formation. In mice injured with a concussion of moderate severity as defined by the 1-h posttraumatic neurologic recovery (grip score), a 60% increase in 2,5-DHBA formation was observed by 1 min after injury compared with that observed in uninjured mice. The peak in DHBA formation occurred at 15 min after injury (+67.5%; p < 0.02, compared with uninjured). At 30 min, the increase in DHBA lost significance, indicating that the posttraumatic increase in brain ^?OH formation is a transient phenomenon. In severely injured mice, the peak increase in DHBA (both 2,3- and 2,5-) was observed at 30 min after injury, but also fell off thereafter as with the moderate injury severity. Preinjury dosing of the mice with SKF-525A (50 mg/kg i.p.), an inhibitor of microsomal drug oxidations, did not blunt the posttraumatic increase in salicylate-derived 2,5-DHBA, thus showing that it is not due to increased metabolic hydroxylation. Neither injury nor SKF-525A administration affected the DHBA plasma levels. However, saline perfusion of the injured mice to remove the intravascular blood before brain removal eliminated the injury-induced increase in 2,5-DHBA, but did not affect the baseline levels seen in uninjured mice. This implies that the source of the increased DHBA in the injured mice is the microvasculature, probably the endothelium. The administration of the 21-aminosteroid lipid antioxidant, tirilazad mesylate, which possesses ^?OH scavenging properties, also attenuated the posttraumatic increase in DHBA, further supporting that it reflects an increase in ^?OH radical formation. These results are the first direct demonstration of the occurrence and time course of increased ^?OH production in injured brain.     

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation

We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (*OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 microl/min. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of *OH. Induction of [K(+)](o) (70 mM) or tyramine (1 mM), significantly increased the formation of *OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K(+) depolarization-induced *OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 microM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K(+) depolarization-induced *OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 microM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced *OH generation by inhibiting LDL oxidation in the heart.     

L-DOPA does not enhance hydroxyl radical formation in the nigrostriatal dopamine system of rats with a unilateral 6-hydroxydopamine lesion

The debate about the toxicity of L-DOPA to dopaminergic neurons has not been resolved. Even though enzymatic and nonenzymatic metabolism of L-DOPA can produce hydrogen peroxide and oxygen free radicals, there has been controversy as to whether L-DOPA generates an oxidant stress in vivo. This study determined whether acute or repeated administration of L-DOPA caused in vivo production of hydroxyl radicals in striatum and other brain regions in rats with a unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal projections. Salicylate trapping combined with in vivo microdialysis provided measurements of extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA) in striatum following L-DOPA administration systemically (100 mg/kg, i.p.) or by intrastriatal perfusion (1 mM, via the microdialysis probe). Tissue concentrations of 2,3-DHBA and salicylate were also measured in striatum, ventral midbrain, and cerebellum following repeated administration of L-DOPA (50 mg/kg, i.p., once daily for 16 days). In each instance, treatment with L-DOPA did not increase 2,3-DHBA concentrations, regardless of the nigrostriatal dopamine system's integrity. When added to the microdialysis perfusion medium, L-DOPA resulted in a significant decrease in the striatal extracellular concentration of 2,3-DHBA. These results suggest that administration of L-DOPA, even at high doses, does not induce hydroxyl radical formation in vivo and under some conditions may actually diminish hydroxyl radical activity. Furthermore, prior damage to the nigrostriatal dopamine system does not appear to predispose surviving dopaminergic neurons to increased hydroxyl radical formation following L-DOPA administration. Unlike L-DOPA, systemic administration of methamphetamine (10 mg/kg, s.c.) produced a significant increase in the concentration of 2,3-DHBA in striatal dialysate, suggesting that increased formation of hydroxyl radicals may contribute to methamphetamine neurotoxicity.     

Aromatic trap analysis of free radicals production in experimental collagen-induced arthritis in the rat: protective effect of glycosaminoglycans treatment

Many findings demonstrated that Glycosaminoglycans (GAGs) and Proteoglycans (PGs) possess antioxidant activity. Collagen-induced arthritis (CIA) is an experimental animal model similar to human rheumatoid arthritis (RA) in which free radicals are involved. Sodium salicylate can be used as a chemical trap for hydroxyl radicals (OH •), the most damaging reactive oxygen species (ROS), yielding 2,5-dihydroxybenzoic acid), (2,5-DHBA) and 2,3-dihydroxybenzoic acid (2,3-DHBA). The measurement of these two acids in the plasma allows to indirectly assess the production of OH • radicals. The aim of the study was to investigate the effect of hyaluronic acid (HYA) (30 mg/kg i.p.) or chondroitin-4-sulphate (C4S) (30 mg/kg i.p.), on free radical production in Lewis rats subjected to CIA. After the immunization with bovine collagen type II in complete Freund's adjuvant, rats developed an erosive hind paw arthritis, that produced high plasma OH • levels assayed as 2,3-DHBA and 2,5-DHBA, primed lipid peroxidation, evaluated by analyzing conjugated dienes (CD) in the articular cartilage; decreased the concentration of endogenous vitamin E (VE) and catalase (CA) in the joint cartilage; enhanced macrophage inflammatory protein-2 (MIP-2) serum levels and increased elastase (ELA) evaluated as an index of activated leukocyte polymophonuclear (PMNs) accumulation in the articular joints. The administration of HYA and C4S starting at the onset of arthritis (day 11) for 20 days, limited inflammation and the clinical signs in the knee and paw, reduced OH • production, decreased CD levels, partially restored the endogenous antioxidants VE and CA, reduced MIP-2 serum levels and limited PMNs infiltration. The results indicate that the GAGs HYA and C4S significantly reduce free radical production in CIA and could be used as a tool to investigate the role of antioxidants in RA.     

Increased generation of hydroxyl radicals in the rat hypertrophied myocardium: in vivo study by microdialysis

A comparative study of the generation of hydroxyl radicals (OH*) in the hypertrophic myocardium of SHR-SP rats (n = 8) and in the myocardium of WKY (n = 5) and Wistar (n = 12) rats was performed using the microdialysis technique. The experiments were carried out on anesthetized open-chest male rats (ketamine intraperitoneally, 10 mg/kg) with artificial ventilation. The amount of OH* produced was estimated by high-performance liquid chromatography with electrochemical detection using as a marker 2,3-dihydroxybenzoic acid (2,3-DHBA), a product of the reaction of the hydroxyl radical with salicylic acid added to the perfusate. The quantity of 2,3-DHBA in the dialysate was estimated by the external standard method and expressed in percent of the 2,3-DHBA concentration in the perfusion fluid. The mean baseline value of 2,3-DHBA in dialysate samples in SHR-SP rats (157 +/- 22%, n = 8) was significantly higher than in Wistar (90 +/- 15%, n = 12, p = 0.0001) and Wistar-Kyoto rats (106 +/- 12%, n = 5, p = 0.005). The basal 2,3-DHBA level in SHR-SP rats was positively correlated (r = 0.831, n = 7, p < 0.05) with the degree of hypertrophy of the left ventricle expressed as the ratio of the left ventricle weight to the body weight. The data presented demonstrate that the hypertrophy of the left ventricle in SHR-SP rats is accompanied by the elevation of the level of free oxygen radicals.     

Protective effect of histidine on iron (II)-induced hydroxyl radical generation in rat hearts.

We investigated the efficacy of histidine on iron (II)-induced hydroxyl radical (.OH) generation in extracellular fluid of the rat myocardium using a flexibly mounted microdialysis technique (O system). Rats were anesthetized and a microdialysis probe was implanted in the left ventricular, followed by infusion of sodium salicylate in Ringer's solution (0.5 nmol/microL/min) to detect the generation .OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Iron (II) clearly produced a concentration-dependent increase in .OH formation. A positive linear correlation between iron (II) and the formation of 2,3-DHBA (R2 = 0.987) was observed. However, histidine (25 mM) was infused through a microdialysis probe; iron (II) failed to increase the 2,3-DHBA formation obtained. To examine the effect of histidine on ischemia-reperfusion of the myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. When corresponding experiments were performed with histidine (25 mM)-pretreated animals, histidine prevented the ischemia-reperfusion induced .OH generation trapped as 2,3-DHBA. These results indicate that histidine protects the myocardium against ischemia-reperfusion damage by .OH generation.     

白藜芦醇对大鼠局灶性脑缺血再灌注的治疗作用

目的:观察白藜芦醇对大鼠局灶性脑缺血再灌注损伤的治疗作用及可能的机制。方法:将SD大鼠随机分为2组:对照组(n=16),白藜芦醇组(n=16)。对照组再灌注即刻腹腔给予0.5 ml生理盐水,白藜芦醇组再灌注即刻腹腔给予20 mg/kg白藜芦醇。再灌注22小时后,进行神经功能学评分、脑梗死容积测定,用分光光度仪测定脑组织溶浆中SOD、MDA和MPO的含量。结果:再灌注22小时后,白藜芦醇治疗组可以改善大鼠神经功能学评分和降低脑梗死面积(P<0.05),同时可以增加脑组织溶浆中SOD的活性,降低MDA和MPO的含量。结论:白藜芦醇通过减轻白细胞的浸润、提高自由基的清除率对大鼠局灶性脑缺血再灌注损伤发挥治疗作用。     

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.