Ischemia/reperfusion damage is an inflammatory process that may arise due to interruption and then subsequent restorage of arterial blood flow due to various reasons. This process may emerge after organ transplantations, vascular operations, or arterial occlusions. During ischemia, xanthine oxidase and hypoxanthine accumulate in the intracellular region due to oxygen deficiency, and ischemia/reperfusion process initiates. Maintenance of the balance between the free oxygen radicals and the protective systems is significant in the generation of ischemia/reperfusion damage.1-4
The degree of reperfusion damage is also related to the duration of ischemia, the number of leukocytes in the area, and the amount of tissue exposed to ischemia.5,6
We have reported before in an experimental study that clopidogrel reduces ischemia reperfusion damage in the skeleton muscle. It was detected in the mentioned study that clopidogrel had a positive effect in preventing reperfusion damage by affecting the malondialdehyde, superoxide dismutase (SOD), and glutathion levels.7
Clopidogrel is known to affect nitric oxide (NO) levels under various conditions. However, there is no published report on the effect of clopidogrel on the NO change occurring during the reperfusion damage.
MATERIALS AND METHODS
This study was conducted in compliance with the guidelines of the National Institutes of Health Guidelines of Care and Use of Laboratory Animals, and approved by the Ethic Committee of Kocaeli University School of Medicine. Male adult Sprague-Dawley rats (250 to 300 g) were used in the study (n = 30). The rats were divided into 3 groups: Control group consisted of rats which were anesthetized with ketamine (n = 10); in ischemia group, the rats were reperfused for 4 hours after ischemia for 6 hours (n = 10) and, in clopidogrel-pretreated group, the rats were pretreated with clopidogrel (Plavix, Sanofi-Synthelabo, Istanbul; Turkey; 0.2 mg/kg/d) for 10 days before ischemia-reperfusion (n = 10). Clopidogrel was administered to the animals via a nasogastric tube. On the eleventh day, circulation of right lower extremity of the animals in groups 2 and 3 was blocked at trochanter major level. Blockade of circulation was confirmed with Doppler ultrasonography (MD 2, Huntleigh Diagnostic Comp, NJ). This method has been shown previously to reduce the blood flow approximately 98% and lead to acute ischemia. After 6 hours of ischemia, the extremity was reperfused for 4 hours. Then, all rats were anesthetized with ketamine (100 mg/kg; Ketalar, Pfizer, Istanbul, Turkey). Blood samples (5 mL) were drawn from the heart and ascending aorta after midsternotomy. Tissue samples were taken from gastrocnemius muscle, liver, and lungs. NO levels were measured in blood and tissue samples.
Tissues were washed in 0.9% NaCl and kept in ice. Tissues were homogenized with cold Tris/HCl buffer (pH 7.4) to make a 10% homogenate (wt/vol). The total of NO-metabolites nitrate (after reduction to nitrite by cadmium granules) + nitrite was assayed colorimetrically using the Griess reaction as described previously. The Griess reagent consists of sulfanilamide and N-(1-naphthyl)ethylenediamine. The method is based on a 2-step process. The first step is the conversion of nitrate to nitrite using nitrate reductase. The second step is the addition of Griess reagent, which converts nitrite into a deep-purple azo compound; photometric measurement of the absorbance at 540 nm due to this azochromophore accurately determines the nitrite concentration (sodium nitrate is used as a standard). Protein interference was eliminated by treatment of the reacted samples with zinc sulfate and centrifugation for 5 minutes at 10,000 g. The results were expressed as μmol/100 mg protein.8
The NO values in 4 different tissues were numerically measured and recorded. Their median, minimum, and maximum values were calculated. To make comparisons between the groups, Kruskall-Wallis analysis of variance was used. On finding statistically significant differences, Bonferroni corrected pair-wise comparisons using Mann-Whitney U test were conducted. Values of P < 0.05 were considered statistically significant. SPSS version 11.5 was used for statistical analysis (Table 1).
NO levels were measured in plasma, lung, liver, and muscle tissues. There were statistically significant differences in the lung tissue and plasma NO measurements among groups (Figs. 1, 2). In addition, the measured NO values for the second group were higher than those in the first and the third groups. In the paired comparisons of level of NO in plasma, although there were no statistically significant differences between groups 1 and 3. There were statistical differences between groups 1 and 2 as well as groups 2 and 3. With respect to lung tissue measurements among groups, there were statistically significant differences among all groups in all paired comparisons. However, there were no statistically significant differences in the muscle and liver tissue NO measurements among groups. The NO levels were higher in the second group in terms of muscle and liver tissue measurements (Figs. 3, 4).
The adenosine 5′ triphosphate reduces in tissues subjected to ischemia. Cell membrane functions are impaired with the reduction in adenosine 5′ triphosphate, which results in an increase in the intracellular calcium level. As a result, calcium-dependent enzymes such as xanthine oxidase and xanthine dehydrogenase are activated. Accumulation of xanthine oxidase and hypoxanthine, on the other hand, triggers various events leading to generation of free oxygen radicals9,10 and intracellular pH decreases. This reduction activates lysosomal lytic enzymes. Hypoxanthine accumulates in the ischemic period, which is converted to xanthine and uric acid in the reperfusion period. Superoxide radicals are generated by the effect of oxygen and xanthine oxidase in the area. Reperfusion also leads to leukocyte activation, adherence, diapedesis, and microvascular dysfunction.11,12 Leukocytes, complements, platelet-activating factor, adhesion molecules, cytokines, microvascular structure, and NO plays important roles in the events occurring during the reperfusion period. NO is a free radical, which has been identified in the recent years and has a very short half-life and significant roles in many biologic events. At first it was identified as an endothelium-derived relaxing factor in vascular system. It is synthesized from L-arginine via the NO synthase (NOS) enzyme. The NOS enzyme exists in 2 main isoforms. One of them is the cNOS (constitutive-NOS) that exists in cells at a constitutively basal level. The other one is the iNOS (inducible-NOS) that is expressed and activated by stimulating factors that take place during some pathologic events. iNOS is synthesized by the stimulation of cytokines, endotoxines, and lipopolysaccharides.
NO synthesized by iNOS is much more in amount compared with cNOS. In addition, the activity of iNOS is longer as it functions independent from calcium and stimulating agents. Therefore, the NO level produced by iNOS is considerably higher than the physiologic limits. NO can be a contributing factor in situations resulting in myocardial function disorder, circulatory failure, and various organ disorders. On the other hand, increased NO can make very favorable contributions to vasodilatation, microcirculation of tissues through prevention of thrombocyte adhesion, and as a result, to the delivery of oxygen to tissues. Although NO production increases directly proportional with the amount of oxygen that exists in the environment, it decreases as a result of endothelial destruction caused by the effects of free oxygen radicals and is blocked as it directly reacts with free oxygen radicals. With the reduction of NO production, vasoactive systems are stimulated, thrombocyte aggregation increases, and endothelial leukocyte adhesion increases. Free oxygen radicals will be formed as a result of leukocyte adhesion. Because the produced NO will react with the free oxygen radicals, the NO level will decrease even further. This leads to the formation of peroxynitrite molecule, which is extremely toxic in the presence of cellular superoxide anion (O2−). Lipid peroxidation, peroxynitrite formation; is depended upon the balance between the production of superoxide and SOD and NO production/utilization. As a result, although NO at lower concentrations regulates cellular functions positively, it may create toxic effects on cells at higher concentrations. If NO and reactive oxygen radicals are produced at equal levels, NO stimulates lipid peroxidation, which causes cellular damage; on the other hand, if more NO is synthesized as compared with reactive oxygen radicals, NO inhibits alcoxyl (RO*) and peroxyl (ROO*) radicals, which reduces peroxidation and protects the cell.13,14
The balance between platelet-activating factor and NO regulates the occurrence of the results induced by hypoxia. Restorage of the blood flow in the ischemic tissue may lead to reperfusion damage. Ischemia/reperfusion damage is the occurrence of cell death owing to inadequate energy sources in ischemia, tissue acidosis, and generation of superoxide radicals after restorage of oxygen in the reperfusion stage. Superoxide radicals cause lipid peroxidation and thus disorder in the cell membrane.15-17
In addition, various antioxidant substances and systems act toward reduction of the superoxide radical effects. Two of these are glutathion and SOD. From time to time, this damage affects far-off organs as well. Even if reperfusion is fully provided, various neurologic, renal or metabolic symptoms may arise in cases. It may lead to impairment of organ functions and loss of the organ, if occurs after organ transplantation in particular.18,19 Development of reperfusion damage symptoms is closely related to the duration of ischemia and to the amount of tissue exposed to ischemia. In the reperfusion period, complements also arise in damaged tissues. These complements influence reperfusion damage by providing chemotaxis and activation of leukocytes.
Mechanical methods and pharmacological agents have been used in the past years to prevent ischemia/reperfusion damage. The pharmacological agents used included vitamin E, flavanoids, mannitol, and ascorbic acid. All the agents used have different action mechanisms.20,21
The aim of our study was the detection of the NO levels in an experimentally created acute skeletal muscle ischemia model, and the investigation of the effect of clopidogrel on these NO levels. It has been detected that NO had protective effects on microvascular structures, inhibiting effects on neutrophil infiltration, and protective effects on vascular permeability. It also can react with free oxygen radicals and has antiplatelet activity. In various studies, leukocyte adhesion to the vascular endothelial cells of subjects who received NO has been detected to be lower.22 Owing to low level of leukocyte penetration, tissue damage is reduced during ischemia reperfusion. Besides this effect, NO reacts with free oxygen radicals to form nitrates, and thus accumulation of hydroxyl radicals is prevented. Furthermore, NO inhibits lipid peroxidation induced by the radicals, by retaining and inhibiting the hydroxyl radicals.23
Measuring NO concentration is very difficult due to its short half-life. Indirect measurement methods are used, which include measurement of the NO metabolites nitrites and nitrates. The role of NO during ischemia reperfusion has not been clearly defined, as its measurement is difficult. Although its beneficial effects were mentioned in some studies, other studies have reported cytotoxic effects.1,22,24-27
Clopidogrel, the agent used in this study, is a thienopyridine group drug, which inhibits binding of adenosine diphosphate (ADP) to the ADP receptors on the platelets. This way, activation of GP IIb/IIIa complex by ADP is selectively inhibited.28
Thrombocyte aggregation and activation are also inhibited. This inhibition is irreversible, and blocks 50% to 70% of the thrombocyte and fibrinogen connections. Clopidogrel is an agent used particularly in chronic arterial ischemias.
It has been reported in previous studies that some members of the thienopyridine group also lead to an increase in NO levels in human neutrophils.29
However, there are very few studies regarding an increase in the NO production induced by clopidogrel.29 We have not found any publications showing the effects of clopidogrel on NO in ischemia/reperfusion events. In a previous report, diabetic retinopathy due to NO deficiency has been created experimentally, after complete recovery of these subjects by thienopyridine therapy. The results of this study also have revealed the effects of the thienopyridine group on NO.30,31
Studies have shown that the correlation between the NO levels and NO effects is not always positive. There are very few studies showing that clopidogrel leads to changes in NO levels. In our study, NO levels were higher in the ischemia group than in the other 2 groups. This difference was statistically significant. The reason that NO levels did not display an increase in our study may be due to the effect of the metabolic substances generated during reperfusion period on NO producing sources. Or the structure and sensitivities of the NO producing sources may have changed during the ischemia and reperfusion stage.
NO level may also have been reduced because the cells providing iNOS activation may have been damaged due to ischemia or reperfusion.
It has been reported in the previously published studies that increased NO levels inhibited platelet adhesion, leukocyte infiltration, and the harmful effects of the superoxide radicals; and had protective effects on vascular permeability. However, NO level was detected to be low in our study in the control and clopidogrel groups. In other words, clopidogrel presented its inhibiting effect on reperfusion damage by reduction in NO levels.
The effects of clopidogrel revealed in this study suggest the need for further studies.
Previous studies have reported on clopidogrel's inhibiting effect on reperfusion damage. However, while inhibiting reperfusion damage, it unusually displays a decreasing effect on the NO level. Perhaps the reperfusion inhibiting effect of clopidogrel is provided only by influencing particular NOS enzyme groups. Thus, through the identification of all the groups of synthase individually, the reduction mechanism of NO will be possible to be identified, and its effect on the reperfusion damage will be defined clearly.
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