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.     

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.     

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.     

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.     

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.     

Hydroxyl Radical Formation in Hyperglycemic Rats During Middle Cerebral Artery Occlusion/Reperfusion

Preexisting hyperglycemia is associated with enhanced reperfusion injury in the postischemic rat brain. The goal of this study was to evaluate whether the hyperglycemic exacerbation of brain injury is associated with enhanced generation of hydroxyl radicals in rats subjected to middle cerebral artery occlusion (2 h), followed by reperfusion (2 h). Magnetic resonance images revealed the exacerbation of focal brain injury in hyperglycemic rats. The salicylate trapping method was used in conjunction with microdialysis to continuously estimate hydroxyl radical production by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) during ischemia/reperfusion. In normoglycemic rats, from a mean baseline level of 130 nmol/l, 2,3-DHBA levels surged to peak levels of 194 nmol/l 45 min into ischemia and to 197 nmol/l 15–30 min into the reperfusion period, returning to baseline by 2 h into reperfusion. A similar temporal profile was observed in hyperglycemic rats, except that absolute 2,3-DHBA levels were higher (165 nmol/l at baseline, 317 nmol/l peak during ischemia, 333 nmol/l peak during reperfusion), and levels remained significantly high (p < .05) throughout the reperfusion period. These results suggest that hydroxyl radical is an important contributor to the exacerbation of neuronal and cerebrovascular injury after focal ischemia/reperfusion in hyperglycemic rats.     

Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions

Abstract

Microdialysis was done on 300–400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D –4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 µl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 µl of all samples: 2,3-DHBA = 7.0 ± 2.5 and 2,5-DHBA = 11.3 ± 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.     

Hydroxyl radicals detected via brain microdialysis in rats breathing air and during hyperbaric oxygen convulsions

Microdialysis was done on 300-400 g, awake, male rats with microdialysis probes inserted through guide cannulas into the striatum (Bregma co-ordinates A 0.5, L 2.9, D -4.0 for guide cannulas implanted 5 days previously). Rats were exposed to hyperbaric oxygen (HBO; 6 atm absolute, 5 atm gauge pressure of oxygen with carbon dioxide absorbed by soda lime). Artificial cerebrospinal fluid (CSF) containing 5 mM sodium salicylate was perfused at 1 microl/min and collected over sequential 10 min intervals with rats breathing air, then HBO, and after decompression. Times to convulsions and duration and severity of convulsions were observed and recorded. CSF samples were analyzed for 2,3- and 2,5-dihydroxybenzoic acid (DHBA), reaction products of hydroxyl radicals with salicylate, by HPLC and compared to authentic standards. Recovery of DHBAs was 48% from fluid surrounding microdialysis probes, based on in vitro tests. The average time to the first convulsion was 21 min and rats convulsed an average of 4 times during 40 min in HBO. There were no significant differences in hydroxyl radical production by this protocol during any of the 10 min collection periods in air or HBO (average in pmoles for 10 microl of all samples: 2,3-DHBA = 7.0 +/- 2.5 and 2,5-DHBA = 11.3 +/- 4.1). The failure to detect an increase in hydroxyl radicals in HBO prior to or during convulsions appears valid since each rat served as its own control.     

In vivo monitoring of norepinephrine and hydroxyl free radical generation by ferrous iron in the myocardium with a microdialysis technique

1. We examined in vivo monitoring of norepinephrine and hydroxyl radical generation in rat myocardium with a microdialysis technique. For this purpose, we designed the microdialysis probe holding system which includes loose fixation of the tube and synchronization of the movement of the heart and the probe.2. The hydroxyl free radical (OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA) which can be measured electrochemically in picomole quantity by high performance liquid chromatography (HPLC).3. After probe implantation, norepinephrine concentration of dialysate decreased over the first 150 min and then reached an almost steady level. A positive linear correlation between the ferrous iron and OH formation trapped as 2,3-DHBA (R² = 0.960) and 2,5-DHBA (R² = 0.982) was observed using the microdialysis technique.4. The present results indicate that non-enzymatic oxidation in the extracellular fluid may play a key role in hydroxyl radical generation by ferrous iron.     

Production of hydroxyl free radical by brain tissues in hyperglycemic rats subjected to transient forebrain ischemia

Preischemic hyperglycemia is known to aggravate brain damage resulting from transient ischemia. In the present study, we explored whether this aggravation is preceded by an enhanced formation of reactive oxygen species (ROS) during the early reperfusion period. To that end, normo- and hyperglycemic rats were subjected to 15 min of forebrain ischemia and allowed recovery periods of 5, 15, and 60 min. Sodium salicylate was injected intraperitoneally in a dose of 100 mg/kg, and tissues were sampled during recirculation to allow analyses of salicylic acid (SA) and its hydroxylation products, 2,3- and 2,5-dihydroxybenzoate (DHBA). Tissue sampled from thalamus and caudoputamen in normoglycemic animals failed to show an increase in 2,3- or 2,5-DHBA after 5 and 15 min of recirculation. However, such an increase was observed in the neocortex after 60 min of recirculation, with a suggested increase in the hippocampus as well. Hyperglycemia had three effects. First, it increased 2,5-DHBA in the thalamus and caudoputamen to values exceeding normoglycemic ones after 15 min of recirculation. Second, it increased basal values of 2,5- and total DHBA in the neocortex. Third, it increased the 60-min values for 2,5- and total DHBA in the hippocampus. These results hint that, at least in part, hyperglycemia may aggravate damage by enhancing basal- and ischemia-triggered production of ROS.     

Time course of production of hydroxyl free radical after subarachnoid hemorrhage in dogs

Vasospasm after subarachnoid hemorrhage (SAH) is associated with lipid peroxidation. However, lipid peroxides increase in a delayed fashion after SAH and may be a byproduct of but not a cause of vasospasm. This study correlated vasospasm with hydroxyl free radical and lipid peroxide levels. 24 dogs had baseline cerebral angiography and induction of SAH by 2 injections of blood into the cisterna magna at baseline and 2 days later. Angiography was repeated 4, 7, 10, 14 or 21 days after the first injection (n = 4 per group) and a microdialysis catheter was inserted into the premedullary cistern. Control dogs (n = 4) underwent angiography and microdialysis but not SAH. Salicylic acid, 100 mg/kg, was administered intravenously, and microdialysis fluid was collected and analyzed by high pressure liquid chromatography for 2,3- and 2,5-dihydroxybenzoic acids (DHBA). Malondialdehyde was measured in subarachnoid clot removed from the prepontine cistern and in the basilar artery itself at the time of euthanasia. Significant vasospasm developed 4 to 14 days after SAH. Malondialdehyde levels were significantly elevated in the basilar artery and subarachnoid clot 4 days after SAH (p < 0.0001, ANOVA) but not at other times. 2,5-DHBA levels were significantly greater than control at 4 to 14 days and they peaked at 4 days (p < 0.05, ANOVA). 2,3-DHBA was significantly increased at 4 days after SAH (p < 0.05, ANOVA). There were significant correlations between basilar artery malondialdehyde levels and vasospasm and cerebrospinal fluid 2,5-DHBA levels and vasospasm. These results suggest the presence of hydroxyl free radical after SAH and demonstrate a correlation between such production, as measured by trapping with salicylate, and the early phase of vasospasm. The correlation with vasospasm implicates free radicals and lipid peroxidation in this phase of vasospasm.     

Comparison of salicylate and D-phenylalanine for detection of hydroxyl radicals in chemical and biological reactions

Summary

Hydroxylation of salicylate and D-phenylalanine was measured to test the usefulness of these compounds for hydroxyl radical (HO^?) detection in chemical and biological systems. When HO^? were produced by the photolytic decomposition of hydrogen peroxide, nearly equal amounts of 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were produced from salicylate, with catechol as a minor product. In the photolytic reaction, nearly equal concentrations of p-,m-, and o-tyrosine were formed from D-phenylalanine. When salicylate or D-phenylalanine was present with Fenton reagents or in iron(II) autoxidation systems, the relative proportions of hydroxylated products were similar to those observed after photolysis, although less total products were usually detected. In contrast, when similar experiments were conducted with isolated hepatic microsomes and perfused livers, 2,5-DHBA was the primary product from salicylate, and p-tyrosine was the major product from D-phenylalanine. Cytochrome P-450 enzymes can hydroxylate salicylate to produce 2,5-DHBA, and it is likely that phenylalanine hydroxylase produces most of the p-tyrosine detected in hepatic tissues. Thus, although both salicylate and D-phenylalanine are useful probes for hydroxyl radical formation in chemical systems, hydroxylated products formed from enzymatic reactions complicate interpretation of data from both compounds in vivo.     

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.     

Salicylate as an in vivo free radical trap: studies on ischemic insult to the rat intestine.

Ischemia of rat intestine was induced in vivo by occlusion of the superior mesenteric artery (SMA) for 15 min. Sodium salicylate, 100 mg/kg, given IP, 30 min prior to the ischemic event served as a specific trap for hydroxyl radicals. Portions of the bowel were sequentially isolated and removed--2 min prior to ischemia, 2 min prior to declamping of the SMA, and 10 min following reperfusion. The bowel segments were homogenized in 3% TCA. The homogenate was centrifuged and filtrated through a 0.22 mu filter. The hydroxylation products of salicylate, dihydroxybenzoic acid (DHBA) derivatives, were isolated, identified, and quantified by HPLC coupled with electrochemical detection (ECD). The level of 2,5-DHBA (M +/- SE, ng/g tissue) in the preischemic bowel (N = 21) was 241.8 +/- 10.0. In the ischemic specimen the level of 2,5-DHBA increased significantly to 313.3 +/- 15.5 (p = 0.0129), and remained unchanged in the reperfusion period (322.8 +/- 15.5). The histological examination correlated well with these levels: mild villi damage in the ischemic period with no further exacerbation during the reperfusion period. This study in an in vivo animal model of intestinal ischemia-reperfusion provides direct evidence for the involvement of free radicals during the ischemic insult.     

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.     

Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCE) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats

Hydroxyl free radicals react with salicylate to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Utilizing the technique of high pressure liquid chromatography with electrochemical detection (LCED), it is possible to detect DHBAs at the level of femtomoles. Since salicylate is relatively non-toxic, we have administered it as a trapping agent in a first attempt to examine the use of the LCED method as a sensitive measure of in vivo OH production. Utilizing adriamycin administration as a model to induce oxygen free radical tissue damage, we found that the level of DHBAs present in drug treated rats versus controls was increased 100-fold in heart and muscle, 30-fold in lung, and 3- and 4-fold in brain and blood, respectively. These first observations support the theory that adriamycin induced OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum

To determine if greater amounts of hydroxyl radical (*OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134microg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8micromoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses *OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) *OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed *OH.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

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.