salicylates and Reperfusion-Injury

Ischemia/reperfusion (I/R) is a condition that stimulates an intense inflammatory response. No ideal treatment exists. Triflusal is an antiplatelet salicylate derivative with anti-inflammatory effects. S-adenosylmethionine is a metabolic precursor for glutathione, an endogenous antioxidant. Dextromethorphan is a low-affinity N-methyl-D-aspartate receptor inhibitor. There is evidence that these agents modulate some of the pathways involved in I/R physiopathology. Intestinal I/R was induced in rats by clamping the superior mesenteric artery for 60 minutes, followed by 60 minutes of reperfusion. Rats either received saline or the drugs studied. At the end of the procedure, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), malonaldehyde (MDA), and total antioxidant capacity (TAC) were determined and intestinal morphology analyzed. I/R resulted in tissue damage, serum TNF-alpha and MDA elevations, and depletion of TAC. All drugs showed tissue protection. Only triflusal reduced TNF-alpha levels. All drugs lowered MDA levels, but only triflusal and S-adenosylmethionine maintained the serum TAC.

Topics: Animals; Antioxidants; Dextromethorphan; Intestines; Male; Malondialdehyde; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Reperfusion Injury; S-Adenosylmethionine; Salicylates; Tumor Necrosis Factor-alpha

Triflusal is a fluorinated derivative of acetylsalicylic acid (ASA) with demonstrated antithrombotic activity. Recently, evidence for a neuroprotective effect has been obtained. The aim of this study was to compare the neuroprotective effects of the main metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid, HTB) and the ASA metabolite salicylic acid (SA) in an in vitro model of anoxia-reoxygenation in rat brain slices. Rat brain slices (n=10 per group) were subjected to a period of anoxia followed by 180 min reoxygenation. We measured oxidative stress parameters (lipid peroxidation, glutathione system), prostaglandins (PGE(2)), nitric oxide pathway activity (NO) (nitrites+nitrates, constitutive and inducible NO synthase activity) and LDH efflux, a biochemical marker of cell death. Various concentrations (10, 100 and 1,000 microM) of triflusal, HTB, ASA or SA were tested. Triflusal at 10, 100 and 1,000 microM decreased LDH efflux in rat brain slices after anoxia/reoxygenation by 24%, 35% and 49% respectively. This effect was proportionately greater than that of ASA (0%, 13% and 32%). The results with HTB were similar to those with triflusal, whereas SA showed a greater protective effect than ASA (13%, 33% and 35%). The antioxidant effects of HTB and SA on the biochemical mechanisms of cell damage studied here were also greater than the effects of triflusal and ASA, a finding attributable mainly to the decrease in lipid peroxidation and to the ability of HTB to also increase glutathione levels. The triflusal metabolite reduced inducible NO synthase activity by 18%, 21% and 30%, whereas SA inhibited this activity by 9%, 17% and 23%. Triflusal and HTB led to greater increases in NO synthase than ASA or AS. In conclusion, the metabolite HTB plays an important role in the neuroprotective effect of triflusal, at least in the experimental model of anoxia-reoxygenation tested here.

Topics: Animals; Aspirin; Brain Chemistry; Dinoprostone; Glutathione; Hypoxia, Brain; In Vitro Techniques; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Nerve Tissue Proteins; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Reperfusion Injury; Salicylates; Thiobarbituric Acid Reactive Substances

Ischaemia-reperfusion (I/R) injury is a model system of oxidative stress and a potential anti-cancer therapy. Tumour cytotoxicity follows oxygen radical damage to the vasculature which is modulated by tumour production of the vasoactive agent, nitric oxide (NO.). In vivo hydroxylation of salicylate, to 2,3- and 2,5-dihydroxybenzoate (DHBs), was used to measure the generation of hydroxyl radicals (OH.) following temporary vascular occlusion in two murine tumours (with widely differing capacity to produce NO.) and normal skin. Significantly greater OH. generation followed I/R of murine adenocarcinoma CaNT tumours (low NO. production) compared to round cell sarcoma SaS tumours (high NO. production) and normal skin. These data suggest that tumour production of NO. confers resistance to I/R injury, in part by reducing production of oxygen radicals and oxidative stress to the vasculature. Inhibition of NO synthase (NOS), during vascular reperfusion, significantly increased OH. generation in both tumour types, but not skin. This increase in cytotoxicity suggests oxidative injury may be attenuation by tumour production of NO.. Hydroxyl radical generation following I/R injury correlated with vascular damage and response of tumours in vivo, but not skin, which indicates a potential therapeutic benefit from this approach.

Topics: Adenocarcinoma; Animals; Catalase; Deferoxamine; Endothelium, Vascular; Female; Free Radical Scavengers; Hydroxyl Radical; Hydroxylation; L-Lactate Dehydrogenase; Mammary Neoplasms, Experimental; Mice; Mice, Inbred CBA; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Reperfusion Injury; Salicylates; Salicylic Acid; Sarcoma, Small Cell

Free-radicals are reported to cause the tissue-damage associated with some toxins and diseases, yet there is no suitable method for routine in-vivo monitoring of these species. This paper introduces an in-vivo microdialysis technique in which the hydroxyl radical reacts with salicylate to generate dihydroxybenzoic acids (DHBA) which are measured by HPLC with electrochemical detection. When pargyline, a monoamine oxidase inhibitor, was infused into rat brain, the levels of DHBA increased markedly. When noradrenaline was administered to animals pre-treated with pargyline, DHBA levels increased markedly compared with the group treated with noradrenaline only. When the heart was subjected to 15-min regional ischaemia by occlusion of the left anterior descending coronary artery, levels of DHBA in heart dialysate were unchanged. Electrical stimulation of the stellate ganglion resulted in marked elevation of levels of DHBA the myocardial dialysate. Infusion of Fe2+ into rat liver resulted in increased formation of DHBA. When the intestine was rendered ischaemic for 10, 20 and 30 min, the highest DHBA level was obtained after 10-min ischaemia and the lowest after 30 min. These results confirm that free-radical production might make a major contribution at certain stages in the progression of the injury.

Topics: Animals; Brain; Chromatography, High Pressure Liquid; Electric Stimulation; Gentisates; Hydrogen Peroxide; Hydroxybenzoates; Hydroxyl Radical; Iron; Ischemia; Jejunum; Liver; Male; Microdialysis; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Myocardium; Norepinephrine; Pargyline; Rats; Rats, Wistar; Reperfusion Injury; Salicylates; Superoxides; Sympathetic Nervous System

Selective neuronal cell death in the CA1 pyramidal cells of the hippocampus and neurons of the dorsolateral striatum as a consequence of brain ischemia/reperfusion (IR) can be ameliorated with brain hypothermia. Since postischemic injury is mediated partially by chemical production of reactive oxygen species (ROS), decreased ROS production may be one of the mechanisms responsible for cerebral protection by hypothermia. To determine if ischemic brain temperature alters ROS production, reversible IR was produced in rats by occlusion of both carotid arteries with hemorrhagic hypotension. After 15 min of ischemia, circulation was restored for 60 min. Brain temperature was maintained during ischemia at either 30, 36, or 39 degrees C and kept at 36-37 degrees C after reperfusion. Using cerebral microdialysis, we measured nonenzymatic hydroxylation of salicylate by HPLC with electrochemical detection in the hippocampus. CBF was also compared among the groups during IR. The results were that normothermic animals during reperfusion had persistently increased levels of the salicylate hydroxylation product, 2,3-dihydroxybenzoic acid (2,3-DHBA), reaching 251% of control at 60 min. This increase in 2,3-DHBA production was potentiated after 60 min of reperfusion (406% of control) with ischemic hyperthermia. In hypothermic ischemia, 2,3-DHBA production at 60 min was attenuated to 160% of control. CBF decreased to approximately 5% of baseline value during ischemia, but increased three- to four-fold relative to control in all three groups. Therefore, the effects of ischemic brain temperature on 2,3-DHBA production did not correlate with changes in CBF during IR. We conclude that brain-temperature-related changes in OH.production are readily detected in the rat and decreased ROS generation may contribute to cerebral protection afforded by hypothermia during brain ischemia.

Topics: Analysis of Variance; Animals; Body Temperature; Brain; Cerebral Cortex; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Free Radical Scavengers; Hippocampus; Hydroxybenzoates; Hydroxyl Radical; Hydroxylation; Iron Chelating Agents; Ischemic Attack, Transient; Laser-Doppler Flowmetry; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salicylates; Salicylic Acid

We report the effects of the free radical scavengers, salicylic acid and bifemerane HC1, on the survival of hippocampal neurons in the Mongolin gerbil model of ischemia-reperfusion brain damage. In addition to performing histological examinations, we used the salicylate-trapping method to measure the amounts of hydroxyl radicals generated in the cerebral cortex and the hippocampus. Ischemia-reperfusion significantly reduced the number of neurons in the CA1 region of the hippocampus. The decrease was largely prevented by pretreating the animals with either salicylate, 20 mg/kg, or bifemerane HC1, 25 mg/kg. Administering salicylate to the animals 30 minutes after reperfusion was also effective, but bifemerane HC1 was ineffective when given after reperfusion. Two minutes after post-ischemic reperfusion, 2,3-dihydroxybenzoic acid levels were significantly elevated in the cerebral cortex and the hippocampus, and 2, 5-dihydroxybenzoic acid levels were significantly elevated in the hippocampus. The increase in 2,5-dyhydroxybenzoic acid in the hippocampus was suppressed by bifemerane HC1 given 30 minutes before exposure to ischemia. These results suggest that hydroxyl radicals are generated in the gerbil model of ischemia-reperfusion brain injury, and that salicylate and bifemerane HC1 partially prevent the loss of neurons in the hippocampus. Hydroxyl radical scavengers may be useful in reducing neuronal damage associated with ischemia-reperfusion injury.

Topics: Animals; Benzhydryl Compounds; Brain Ischemia; Cell Survival; Cerebral Cortex; Free Radical Scavengers; Gerbillinae; Hippocampus; Hydroxybenzoates; Hydroxyl Radical; Male; Neurons; Reperfusion Injury; Salicylates; Salicylic Acid

Oxygen free radicals may play a pivotal role in the development of the shock-induced inflammatory response syndrome. Hydroxyl free radicals (.OH) react with salicylate (SA) to form 2,5- and 2,3-dihydroxybenzoic acid (DHBA) products. We utilized salicylate hydroxylation to investigate .OH formation during intestinal ischemia/reperfusion injury in male Sprague-Dawley rats. After administering salicylate, the superior mesenteric artery was occluded for 45 min and then allowed to reperfuse for 90 min after declamping. No significant changes in plasma 2,3- and 2,5-DHBA/SA ratios were observed in sham-operated or in animals given intestinal ischemia without reperfusion. A significant increase (p < .05) in arterial, venous, and portal venous 2,5-DHBA/SA ratios occurred 5 min after reperfusion. This increase was prevented by allopurinol as well as by dimethylthiourea (.OH scavenger) pretreatment. 2,3-DHBA was significantly increased (p < .05) in venous and portal venous blood after 30 min of reperfusion, but was not detectable in plasma of allopurinol- and dimethylthiourea-treated rats. These results indicate that hydroxyl free radical formation as reported by SA hydroxylation appears to be important in intestinal ischemia/reperfusion-related tissue injury.

Topics: Animals; Blood Pressure; Free Radicals; Hydroxylation; Intestines; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salicylates; Salicylic Acid

A previous study indicated that hydroxyl radicals are generated in the cat retina during the early reperfusion phase after 90 minutes of ischemia. Salicylate was injected intravenously, and its conversion to 2,3-dihydroxybenzoic acid (2,3-DHBA) served as a marker of hydroxyl radicals. The authors attempted to prevent this free radical generation during reperfusion.. After salicylate administration, both eyes (15 minutes apart) of 15 cats were subjected to 90 minutes retinal ischemia. Following 5 minutes of reperfusion in the control eye, it was enucleated and processed for DHBA quantitation. Then, 7.5 mg of Zn-desferrioxamine (Zn-DFO) was injected intravenously into nine cats and saline into six cats. Five minutes later, reperfusion was induced in the experimental eye for 5 minutes, followed by enucleation. In one eye each of 12 other cats, scotopic electroretinographic (ERG) studies were carried out during 90 minutes of ischemia and 16 to 18 hours of reperfusion. Five minutes before termination of the ischemia, six animals were injected with 7.5 mg Zn-DFO and six with saline.. The normalized levels of 2,3-DHBA were lower in the experimental eyes than in their fellow controls only after Zn-DFO treatment (P = 0.01). In the ERG studies, after 16 to 18 hours of reperfusion, the mean b-wave-ERG amplitudes in the eyes of the saline-treated cats (n = 6) were 8.4% +/- 4.0% of the preischemic stage, and 70.5% +/- 6.7% of the Zn-DFO-treated cats (n = 6, P = 0.004).. Protection of the cat retina against ischemia and reperfusion injury by Zn-DFO was evident, most probably through its inhibitory effect on the generation of hydroxyl radicals during reperfusion.

Topics: Animals; Cats; Deferoxamine; Electroretinography; Fundus Oculi; Hydroxybenzoates; Hydroxyl Radical; Injections, Intravenous; Ischemia; Organometallic Compounds; Reperfusion Injury; Retinal Vessels; Salicylates; Salicylic Acid

Recent studies have shown the ability of salicylic acid (SA) to trap hydroxyl radicals (OH.) generated during reperfusion in ischemic myocardium. Since OH. is implicated in the pathogenesis of reperfusion injury, we examined the effect of SA on reperfusion-induced arrhythmias and postischemic ventricular dysfunction. Isolated rat hearts perfused by the Langendorff technique were preperfused with Krebs-Henseleit buffer containing SA for 10 min. Hearts were then made ischemic for 30 min, followed by 30 min of reperfusion. In a separate group, SA was administered only at the onset of reperfusion. The left ventricular contractile functions, left ventricular developed pressure (LVDP) and its first derivative (LV dP/dt), coronary flow (CF), and creatine kinase (CK) release were determined before and after ischemia. Epicardial electrocardiogram (ECG) was also recorded to analyze the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF). SA improved LVDP, LV dp/dt, and CF recovery and reduced CK release compared to the control group. The incidence of VT and VF during reperfusion was also significantly reduced by SA. Analysis of tissue thiobarbituric acid-reactive products indicates that SA decreased oxidative stress during reperfusion. In conclusion, these results suggest that SA reduces myocardial reperfusion injury and attenuates ventricular arrhythmias by trapping OH. radicals upon reperfusion in isolated rat hearts.

Topics: Animals; Arrhythmias, Cardiac; Creatine Kinase; Electrocardiography; In Vitro Techniques; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Salicylates; Salicylic Acid; Ventricular Function, Left

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.

Topics: Animals; Disease Models, Animal; Free Radicals; Gentisates; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Intestines; Male; Rats; Reperfusion Injury; Salicylates; Salicylic Acid

Brain is extremely susceptible to oxidative damage. Utilizing a series of novel approaches, we have demonstrated that oxidative damage occurs during an ischemia/reperfusion insult (IRI) to brain. Thus, we have demonstrated that an IRI to Mongolian gerbil brain results in: (1) an enhanced rate of salicylate hydroxylation, implicating an increased flux of hydroxyl free radicals; (2) an enhanced flux of free radicals as determined by spin-trapping; (3) an enhanced level of endogenous protein oxidation; (4) a decrease in glutamine synthetase (GS) activity, an enzyme very sensitive to oxidative damage; and (5) demonstration of protection from an IRI by administering the spin-trapping agent alpha-phenyl-tert-butyl nitrone (PBN). The novel observation that PBN offers protection from the lethality brought on by a brain IRI appears to be clearly linked to the ability of the administered spin-trap to inhibit oxidative damage as evidenced by the decreased amount of brain protein oxidation and the prevention of an IRI-mediated loss of GS activity in treated animals. Aged gerbils are more sensitive to the lethal action of a brain IRI than younger animals, but they are protected by PBN administration as are the younger animals. Older gerbils have a significantly higher level of oxidized protein in the brain. Older gerbils have decreased activities of GS and neutral protease, the enzyme that removes oxidized protein, than younger animals. Chronic twice daily administration of PBN (32 mg/kg) for 14 days to older animals significantly lowered brain oxidized protein levels and raised GS and neutral protease activity to those observed in younger animals. Cessation of PBN administration resulted in a time-dependent restoration of protein oxidation levels and enzyme activities back to those observed prior to spin-trap administration. Older gerbils exhibit significantly higher errors in a radial arm maze than younger animals, but older gerbils that had received chronic daily treatments of PBN (32 mg/kg) for 14 days committed significantly less errors than untreated controls. The errors committed in PBN-treated animals was decreased down to the level of those observed in younger animals. Clearly the spin-trapping agent, PBN, appears to have promise in: (1) elucidation of the role of oxidative damage in normal brain function during aging, (2) understanding the development of pathological conditions, and (3) development of treatment regimens for prevention of damage that occurs d

Topics: Animals; Brain; Cerebral Cortex; Cyclic N-Oxides; Free Radicals; Gerbillinae; Glutamate-Ammonia Ligase; Ischemic Attack, Transient; Nitrogen Oxides; Oxidation-Reduction; Oxygen; Reperfusion Injury; Salicylates; Salicylic Acid; Spin Labels

Salicylic acid was used as a probe for .OH formed during reperfusion of the ischemic myocardium. .OH adds to the phenolic ring of salicylate to yield dihydroxybenzoic acid species. The two principal dihydroxybenzoic acids formed are the 2,3- and 2,5-derivatives and can be isolated and quantitated using HPLC combined with electrochemical detection. In these experiments, dihydroxybenzoic acids were detectable in the f molar range. Rat hearts were perfused in the Langendorff mode with Krebs-Henseleit buffer containing 100 microM salicylate. Following 20 min of global ischemia a 173% increase in tissue content of 2,5-dihydroxybenzoic acid was detected after 2.5 min of reperfusion. The duration of ischemia did not significantly affect tissue content of 2,5-dihydroxybenzoic acid peaked at 250 to 300% of control within 2.5 min of reperfusion. The inclusion of 100 microM salicylate in the perfusion buffer had no effect on myocardial function during the duration of the experiments. The results indicate that salicylate can be used as a very sensitive probe for .OH in the isolated ischemic heart.

Topics: Animals; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Free Radicals; Hydroxides; Hydroxybenzoates; Hydroxyl Radical; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Salicylates; Salicylic Acid