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the chief hormone of the pineal gland&#44; is a well-known potent antioxidant and free radical scavenger<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">10&#44;11</span></a> that can counteract the damaging effects of free radicals&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">12&#44;13</span></a> Melatonin also shows a protective effect on lung injuries induced by ischemia&#8211;reperfusion&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">14</span></a> There have been no studies to test the effect of melatonin on lung histological changes induced by skeletal muscle ischemia&#8211;reperfusion&#46; The aim of this study was to examine the effects of melatonin on acute lung injury in a rat model of skeletal muscle ischemia&#8211;reperfusion by transient femoral artery occlusion&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Materials and methods</span><p id="par0015" class="elsevierStylePara elsevierViewall">All experimental procedures were performed according to the guidelines for the ethical treatment of experimental animals and approved by Islamic Azad University School of Veterinary Science&#44; Animal Care and Use Local Ethics Committee&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Experimental groups</span><p id="par0020" class="elsevierStylePara elsevierViewall">Thirty male Wistar rats weighing 300<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>35<span class="elsevierStyleHsp" style=""></span>g were used in this study&#46; They were maintained under constant room temperature of 25<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; relative humidity of 75<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#37;&#44; 12<span class="elsevierStyleHsp" style=""></span>h&#47;12<span class="elsevierStyleHsp" style=""></span>h light&#47;dark cycle&#44; with <span class="elsevierStyleItalic">ad libitum</span> access to water and commercial food and were placed in individual cages&#46; Animals were randomly allocated into three experimental groups of ten rats each&#58;</p><p id="par0025" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group I</span> &#8211; Sham group with no ischemia&#8211;reperfusion&#46; The animals were subjected to all operative procedures&#44; except arterial occlusion and reperfusion&#46; After isolation and exposure of the femoral artery for 2<span class="elsevierStyleHsp" style=""></span>h&#44; the animals received 2<span class="elsevierStyleHsp" style=""></span>ml of 0&#46;9&#37; saline via the external jugular vein&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group II &#40;ischemia&#8211;reperfusion group&#41;</span> &#8211; The animals were subjected to 2<span class="elsevierStyleHsp" style=""></span>h of ischemia followed by 24<span class="elsevierStyleHsp" style=""></span>h of reperfusion&#46; Two milliliters of 0&#46;9&#37; saline was administered intravenously prior to reperfusion&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group III &#40;ischemia&#8211;reperfusion</span><span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">melatonin group&#41;</span> &#8211; All animals underwent 2<span class="elsevierStyleHsp" style=""></span>hours of ischemia by occlusion of the femoral artery followed by 24<span class="elsevierStyleHsp" style=""></span>h of reperfusion&#46; A solution of 10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg melatonin was administered immediately via the external jugular vein&#44; with a total volume of 2<span class="elsevierStyleHsp" style=""></span>ml&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Surgery</span><p id="par0040" class="elsevierStylePara elsevierViewall">Anesthesia was induced using ketamine plus xylazine &#40;10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg and 50<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;m&#46;&#44; respectively&#41;&#46; After induction of anesthesia&#44; the left hind limb was prepared for sterile surgery&#46; A skin incision was made on the medial surface of the left hind limb and the femoral artery was isolated and clamped with a vascular forceps for 2<span class="elsevierStyleHsp" style=""></span>h&#46; Following the ischemic period&#44; the vascular forceps was removed and then the surgical site was routinely closed&#46; Rats were returned to their cages with a commercial balanced diet and water <span class="elsevierStyleItalic">ad libitum</span>&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Specimen collection</span><p id="par0045" class="elsevierStylePara elsevierViewall">At the end of the trial&#44; the rats were euthanized with an overdose of intraperitoneal pentobarbital injection &#40;300<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#41; and the lungs were removed <span class="elsevierStyleItalic">en bloc</span>&#46; The right lungs were used for water content determination and histopathological analysis under light microscope and the left lungs were stored at &#8722;20<span class="elsevierStyleHsp" style=""></span>&#176;C till the biochemical analysis&#46; The lung tissue homogenate and supernatant samples were prepared as described by Yildirim et al&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">15</span></a></p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Biochemical assay</span><p id="par0050" class="elsevierStylePara elsevierViewall">Myeloperoxidase &#40;MPO&#41; 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The ratio of lung wet weight to dry weight was then calculated&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Histopathological examination</span><p id="par0060" class="elsevierStylePara elsevierViewall">The anterior lobe of the right lung &#40;tissue&#41; was placed in a 10&#37; formalin solution and processed routinely by embedding in paraffin and then tissues were sectioned into 5<span class="elsevierStyleHsp" style=""></span>&#956;m pieces and stained with hematoxylin &#38; eosin stain&#46; Pulmonary injury was graded into four grades as follows&#58; grade 0&#44; no diagnostic change&#59; grade 1&#44; mild neutrophil leukocyte infiltration and mild to moderate interstitial congestion&#59; grade 2&#44; moderate neutrophil leukocyte infiltration&#44; perivascular edema formation&#44; and partial destruction of pulmonary architecture&#59; and grade 3&#44; dense neutrophil leukocyte infiltration and complete destruction of pulmonary structure&#46;<a class="elsevierStyleCrossRef" href="#bib0315"><span class="elsevierStyleSup">19</span></a> A total of five slides from each lung sample were randomly screened&#44; and the mean was accepted as the representative value of the sample&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">The Statistical Package for the Social Sciences for Windows 11&#46;5 was used for statistical analysis&#46; All values were given as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; Differences between groups were statistically analyzed by Kruskal&#8211;Wallis test &#40;ANOVA&#41; and chi-square test&#46; Statistical significance was accepted as <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Results</span><p id="par0070" class="elsevierStylePara elsevierViewall">All of the rats survived until the end of the study period&#46; Data belonging to histological changes from lung samples after reperfusion are represented in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#46; In the ischemia&#8211;reperfusion group&#44; tissues showed histological changes with partial destruction of pulmonary architecture&#44; neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46; Grades 1&#44; 2 and 3 of lesions were obtained in the ischemia&#8211;reperfusion group&#44; with an average of 2&#46;3&#46; These pathological changes in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; particularly neutrophilic infiltration and edema&#44; were much less than in the ischemia&#8211;reperfusion group &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Four animals in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group presented no injury and the others had lesions grades 1 and 2&#44; averaging 0&#46;7&#46; Histopathologically&#44; there was a significant difference &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; between the two groups&#46; The average of histological changes in the sham group &#40;0&#46;2&#41; was lower than the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; but with no statistical significance &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0075" class="elsevierStylePara elsevierViewall">Measurement of lung water content was used to estimate pulmonary edema&#46; Compared with the ischemia&#8211;reperfusion group&#44; the lung water content in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups was decreased significantly &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41;&#46; No significant difference in lung wet&#47;dry weight ratio was found between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">The NO level of lung tissue was increased in the ischemia&#8211;reperfusion group &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; The treatment with melatonin recovered this increase and caused a reduction in comparison with the ischemia&#8211;reperfusion group &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41;&#46; No significant difference in NO level was found between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">The novel indicator for neutrophil function&#44; MPO activity&#44; in the ischemia&#8211;reperfusion group was found to be increased dramatically compared to the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; MPO activity comparison between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups did not show significant results &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Discussion</span><p id="par0090" class="elsevierStylePara elsevierViewall">Peripheral arterial clamping&#44; as a surgical procedure&#44; is routinely used for orthopedic surgery or trauma in elective and emergency cases&#46; Lung damage has been shown to occur following transient arterial occlusion&#46; The ischemic damage resulted from a decrease in the blood flow to an organ&#46; When restoring blood flow a more pronounced damage&#44; so called reperfusion injury&#44; occurs&#46; In the development of ischemia&#8211;reperfusion injury&#44; the enhanced generation of oxygen radicals has been suggested&#46;<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">20</span></a> Free radicals have been previously demonstrated to play a significant role in the etiology of remote lung injury in animal models&#46;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">4&#44;21</span></a> It has been widely described that lung injury often occurs after skeletal muscle ischemia due to release of free radicals of reperfusing tissues&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">4</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Melatonin and its metabolites have been shown to have antioxidant properties in <span class="elsevierStyleItalic">in vitro</span> and <span class="elsevierStyleItalic">in vivo</span> systems&#44; both in cells and body fluids&#46;<a class="elsevierStyleCrossRefs" href="#bib0330"><span class="elsevierStyleSup">22&#8211;24</span></a> Furthermore&#44; melatonin plays an important role in activating antioxidant defences&#44;<a class="elsevierStyleCrossRefs" href="#bib0345"><span class="elsevierStyleSup">25&#44;26</span></a> and protects cells from oxidative loads and apoptosis induced by mitochondrial DNA deletion&#46;<a class="elsevierStyleCrossRef" href="#bib0355"><span class="elsevierStyleSup">27</span></a> Both effects allow melatonin to reduce the extent of reactive oxygen species&#44; improving the outcomes of oxidative related pathologies such as ischemia<a class="elsevierStyleCrossRef" href="#bib0395"><span class="elsevierStyleSup">35</span></a> and prevention of aging&#46;<a class="elsevierStyleCrossRefs" href="#bib0360"><span class="elsevierStyleSup">28&#8211;30</span></a>It has been demonstrated that melatonin can prevent ischemia&#8211;reperfusion injury in cerebral artery occlusion induced stroke&#44; in traumatic brain and spinal cord injuries&#44; in animal models&#46;<a class="elsevierStyleCrossRefs" href="#bib0375"><span class="elsevierStyleSup">31&#8211;33</span></a> Besides the generation of free radicals&#44; the ischemia&#8211;reperfusion injury is associated with exaggerated inflammatory response&#46;<a class="elsevierStyleCrossRef" href="#bib0390"><span class="elsevierStyleSup">34</span></a> Activated polymorphonuclear leukocytes aggregate and adhere to endothelium&#44; and impair blood flow and development of endothelial cell edema&#46;<a class="elsevierStyleCrossRef" href="#bib0395"><span class="elsevierStyleSup">35</span></a> Recently&#44; melatonin reduced morphological injury due to inhibition of neutrophils infiltration into the reperfused intestine with abolished P-selectin expression and intercellular adhesion molecule-1 up-regulation on endothelial cells&#46;<a class="elsevierStyleCrossRef" href="#bib0380"><span class="elsevierStyleSup">32</span></a></p><p id="par0100" class="elsevierStylePara elsevierViewall">Several methods have been used to define the role of neutrophils in reperfusion tissue injury&#46; MPO plays a fundamental role in oxidant production by neutrophils&#46; Neutrophils are a main potential source of oxygen-free radicals&#46;<a class="elsevierStyleCrossRef" href="#bib0400"><span class="elsevierStyleSup">36</span></a> A previous study clearly demonstrated that skeletal muscle ischemia&#8211;reperfusion led to remote lung inflammation&#44; which was characterized by increased MPO activity and significant neutrophil infiltration in the lung&#46;<a class="elsevierStyleCrossRef" href="#bib0405"><span class="elsevierStyleSup">37</span></a> NO level is a significant determinant of the lung injury process which is caused by lower extremity ischemia&#8211;reperfusion&#46;<a class="elsevierStyleCrossRef" href="#bib0410"><span class="elsevierStyleSup">38</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">The effects of melatonin on ischemia&#8211;reperfusion injury in an <span class="elsevierStyleItalic">ex vivo</span> rat heart model was studied by Lagneux et al&#46;<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">39</span></a> The authors explained that the melatonin significantly reduced infarct size and the incidence of reperfusion arrhythmias&#46;<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">39</span></a> In an experimental study Erkanl&#305; et al&#46;<a class="elsevierStyleCrossRef" href="#bib0420"><span class="elsevierStyleSup">40</span></a> reported that melatonin has a protective effect against skeletal muscle ischemia&#8211;reperfusion injury and can reduce the incidence of compartment syndrome after chronic or acute peripheral arterial occlusions&#46; Other studies have shown that melatonin protects kidney&#44;<a class="elsevierStyleCrossRef" href="#bib0425"><span class="elsevierStyleSup">41</span></a> liver&#44;<a class="elsevierStyleCrossRef" href="#bib0430"><span class="elsevierStyleSup">42</span></a> testis<a class="elsevierStyleCrossRef" href="#bib0435"><span class="elsevierStyleSup">43</span></a> and intestine<a class="elsevierStyleCrossRef" href="#bib0440"><span class="elsevierStyleSup">44</span></a> against ischemia&#8211;reperfusion injury&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">In the last few years&#44; Okutan et al&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">14</span></a> investigated the protective role of melatonin in lung remote organ injury&#46; For that purpose&#44; they induced lung injury by the occlusion of infrarenal abdominal aorta for 30<span class="elsevierStyleHsp" style=""></span>min and reperfusion for 12<span class="elsevierStyleHsp" style=""></span>h&#46; These authors administered melatonin intraperitoneally at 20<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#44; 1<span class="elsevierStyleHsp" style=""></span>h prior to trial&#46; They found that pre-treating rats with melatonin significantly decreases malondialdehyde &#40;MDA&#41; levels as well as MPO activity&#46; Based on this finding&#44; they believed that their study indicated the potential of attenuating lung injury&#44; and require further studies to show improvement in the morphology of the injured lungs&#46; In the current research&#44; we tested the hypothesis that 10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;v&#46; of melatonin could protect the lungs after 2<span class="elsevierStyleHsp" style=""></span>h femoral artery occlusion and 24<span class="elsevierStyleHsp" style=""></span>h reperfusion&#46; In this study melatonin inhibited MPO activity and decreased NO level which was increased by ischemia&#8211;reperfusion&#46; Pulmonary architecture&#44; neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema were much reduced in the treated group&#46; However&#44; there are further studies to be conducted to test the effects of various doses of melatonin treatment against ischemia&#8211;reperfusion injury in lung&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">Sharma et al&#46;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">18</span></a> showed that lungs undergoing ischemia&#8211;reperfusion displayed increased vascular permeability and pulmonary edema&#46; The conclusion of increased vascular permeability is an assumption based on the lung wet&#92;dry weight ratio&#46; Significant lung edema occurred after ischemia&#8211;reperfusion in rat which was attenuated by melatonin treatment&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">Data acquired demonstrates that melatonin can significantly decrease the severity of pulmonary edema&#44; decrease vascular permeability&#44; decrease leukocytes infiltration and inhibits cellular apoptosis&#44; after skeletal muscle ischemia&#8211;reperfusion&#46; Our findings support the potential use of melatonin as a therapeutic agent in the prevention of acute lung injury associated with skeletal muscle ischemia&#8211;reperfusion&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Ethical disclosures</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Protection of human and animal subjects</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association &#40;Declaration of Helsinki&#41;&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Confidentiality of data</span><p id="par0130" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Right to privacy and informed consent</span><p id="par0135" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Conflicts of interest</span><p id="par0140" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare&#46;</p></span></span>"
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          "titulo" => "Introduction"
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          "titulo" => "Surgery"
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          "titulo" => "Specimen collection"
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          "titulo" => "Biochemical assay"
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          "titulo" => "Lung wet&#47;dry weight ratio"
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          "titulo" => "Histopathological examination"
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              "titulo" => "Protection of human and animal subjects"
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    "fechaRecibido" => "2013-09-10"
    "fechaAceptado" => "2014-01-08"
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          "palabras" => array:6 [
            0 => "Melatonin"
            1 => "Ischemia&#8211;reperfusion"
            2 => "Lung remote injury"
            3 => "Histopathology"
            4 => "Myeloperoxidase"
            5 => "Nitric oxide"
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    "resumen" => array:1 [
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">This study evaluated the protective antioxidant effect of melatonin on lung injury as a remote organ after skeletal muscle ischemia&#8211;reperfusion in rats&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Thirty male Wistar rats were allocated randomly into three experimental groups&#58; operated with no ischemia &#40;Sham&#41; group&#44; ischemia&#8211;reperfusion group and ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#46; Hind limb ischemia was induced by clamping the femoral artery&#46; After 2<span class="elsevierStyleHsp" style=""></span>h ischemia&#44; the clamp was removed and the animal underwent 24<span class="elsevierStyleHsp" style=""></span>h reperfusion&#46; Rats in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group received melatonin &#40;10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;v&#46;&#41;&#44; immediately before the clamp was removed&#46; At the end of the trial&#44; animals were euthanized and the lungs were removed for water content determination&#44; histopathological and biochemical studies&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">In the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; tissues showed less intense histological abnormalities such as neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema compared with the ischemia&#8211;reperfusion group&#46; Histopathologically&#44; there was a significant difference &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; between the two groups&#46; The lung water content in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group was significantly lower than the ischemia&#8211;reperfusion group &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Lung tissue myeloperoxidase &#40;MPO&#41; activity and nitric oxide &#40;NO&#41; level were significantly &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; increased by ischemia&#8211;reperfusion&#46; The increase in these parameters was reduced by melatonin&#46;</p><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Comparing the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group with the sham group&#44; no significant increase in all analyzed aspects of the research was observed&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">These findings suggest that melatonin has preventive effects in lung tissue injury after transient femoral artery occlusion&#46;</p></span>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Groups&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Histological score&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">MPO &#40;U&#47;g protein&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">NO &#40;&#956;mol&#47;g protein&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Lung wet&#47;dry weight&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top">Sham&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top">Ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;17<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">&#43;</span></a>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">5&#46;01<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;39<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">&#43;</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr></tbody></table>
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Histopathologic evaluation&#44; myeloperoxidase &#40;MPO&#41; activity and level of nitric oxide &#40;NO&#41; in lung tissue for each group&#46;</p>"
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Original article
Melatonin attenuates lung injury in a hind limb ischemia–reperfusion rat model
Hamed Takhtfooladia, Mohammad Takhtfooladib,
Corresponding author
dr_ashrafzadeh@yahoo.com

Corresponding author.
, Fariborz Moayerc, Sayed Mobarakehd
a Faculty of Veterinary Science, Karaj Branch, Islamic Azad University, Karaj, Iran
b Department of Surgery, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
c Department of Pathobiology, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran
d Faculty of Medical Sciences, Shahid Sadughi University, Yazd, Iran
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    "titulo" => "Melatonin attenuates lung injury in a hind limb ischemia&#8211;reperfusion rat model"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Light microscopic view of lung tissues from ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#46; More preservation of nearly normal structure &#40;magnification of 10<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10&#44; H&#38;E staining&#41;&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">The pathophysiology of ischemia&#8211;reperfusion injury has been previously described&#46; Polymorphonuclear leukocytes and free radical species have been shown to have pivotal roles in the etiology&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">1</span></a> Skeletal muscle ischemia&#8211;reperfusion resulting from trauma&#44; limb revascularization&#44; orthopedic surgery and free-flap reconstruction or any other condition not only leads to local damage&#44; but also causes severe injury involving destruction of remote organs&#46;<a class="elsevierStyleCrossRefs" href="#bib0230"><span class="elsevierStyleSup">2&#8211;4</span></a>Many studies have shown that several agents such as caffeic acid phenethyl ester or erdosteine&#44;<a class="elsevierStyleCrossRefs" href="#bib0245"><span class="elsevierStyleSup">5&#44;6</span></a> N-acetylcysteine&#44;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">4</span></a> zinc aspartate&#44;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">7</span></a> trapidil<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">8</span></a> and tramadol<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">9</span></a> are useful against lung injury induced by oxidative stress damage in ischemia&#8211;reperfusion&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Melatonin&#44; the chief hormone of the pineal gland&#44; is a well-known potent antioxidant and free radical scavenger<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">10&#44;11</span></a> that can counteract the damaging effects of free radicals&#46;<a class="elsevierStyleCrossRefs" href="#bib0280"><span class="elsevierStyleSup">12&#44;13</span></a> Melatonin also shows a protective effect on lung injuries induced by ischemia&#8211;reperfusion&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">14</span></a> There have been no studies to test the effect of melatonin on lung histological changes induced by skeletal muscle ischemia&#8211;reperfusion&#46; The aim of this study was to examine the effects of melatonin on acute lung injury in a rat model of skeletal muscle ischemia&#8211;reperfusion by transient femoral artery occlusion&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Materials and methods</span><p id="par0015" class="elsevierStylePara elsevierViewall">All experimental procedures were performed according to the guidelines for the ethical treatment of experimental animals and approved by Islamic Azad University School of Veterinary Science&#44; Animal Care and Use Local Ethics Committee&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Experimental groups</span><p id="par0020" class="elsevierStylePara elsevierViewall">Thirty male Wistar rats weighing 300<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>35<span class="elsevierStyleHsp" style=""></span>g were used in this study&#46; They were maintained under constant room temperature of 25<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>2<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; relative humidity of 75<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#37;&#44; 12<span class="elsevierStyleHsp" style=""></span>h&#47;12<span class="elsevierStyleHsp" style=""></span>h light&#47;dark cycle&#44; with <span class="elsevierStyleItalic">ad libitum</span> access to water and commercial food and were placed in individual cages&#46; Animals were randomly allocated into three experimental groups of ten rats each&#58;</p><p id="par0025" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group I</span> &#8211; Sham group with no ischemia&#8211;reperfusion&#46; The animals were subjected to all operative procedures&#44; except arterial occlusion and reperfusion&#46; After isolation and exposure of the femoral artery for 2<span class="elsevierStyleHsp" style=""></span>h&#44; the animals received 2<span class="elsevierStyleHsp" style=""></span>ml of 0&#46;9&#37; saline via the external jugular vein&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group II &#40;ischemia&#8211;reperfusion group&#41;</span> &#8211; The animals were subjected to 2<span class="elsevierStyleHsp" style=""></span>h of ischemia followed by 24<span class="elsevierStyleHsp" style=""></span>h of reperfusion&#46; Two milliliters of 0&#46;9&#37; saline was administered intravenously prior to reperfusion&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Group III &#40;ischemia&#8211;reperfusion</span><span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">melatonin group&#41;</span> &#8211; All animals underwent 2<span class="elsevierStyleHsp" style=""></span>hours of ischemia by occlusion of the femoral artery followed by 24<span class="elsevierStyleHsp" style=""></span>h of reperfusion&#46; A solution of 10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg melatonin was administered immediately via the external jugular vein&#44; with a total volume of 2<span class="elsevierStyleHsp" style=""></span>ml&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Surgery</span><p id="par0040" class="elsevierStylePara elsevierViewall">Anesthesia was induced using ketamine plus xylazine &#40;10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg and 50<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;m&#46;&#44; respectively&#41;&#46; After induction of anesthesia&#44; the left hind limb was prepared for sterile surgery&#46; A skin incision was made on the medial surface of the left hind limb and the femoral artery was isolated and clamped with a vascular forceps for 2<span class="elsevierStyleHsp" style=""></span>h&#46; Following the ischemic period&#44; the vascular forceps was removed and then the surgical site was routinely closed&#46; Rats were returned to their cages with a commercial balanced diet and water <span class="elsevierStyleItalic">ad libitum</span>&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Specimen collection</span><p id="par0045" class="elsevierStylePara elsevierViewall">At the end of the trial&#44; the rats were euthanized with an overdose of intraperitoneal pentobarbital injection &#40;300<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#41; and the lungs were removed <span class="elsevierStyleItalic">en bloc</span>&#46; The right lungs were used for water content determination and histopathological analysis under light microscope and the left lungs were stored at &#8722;20<span class="elsevierStyleHsp" style=""></span>&#176;C till the biochemical analysis&#46; The lung tissue homogenate and supernatant samples were prepared as described by Yildirim et al&#46;<a class="elsevierStyleCrossRef" href="#bib0295"><span class="elsevierStyleSup">15</span></a></p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Biochemical assay</span><p id="par0050" class="elsevierStylePara elsevierViewall">Myeloperoxidase &#40;MPO&#41; activity and nitric oxide &#40;NO&#41; levels were studied in lung tissues&#46; The activity of MPO was analyzed spectrophotometrically as described by Wei et al&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">16</span></a> NO level in lung tissue was measured by Griess reaction&#46;<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">17</span></a></p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Lung wet&#47;dry weight ratio</span><p id="par0055" class="elsevierStylePara elsevierViewall">Lung wet&#47;dry weight ratio was used as an indicator of pulmonary edema&#46; The lower lobe of the right lung from each animal was weighed and placed in a vacuum oven &#40;at 54<span class="elsevierStyleHsp" style=""></span>&#176;C&#41; until a stable&#44;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">18</span></a> dry weight was achieved&#46; The ratio of lung wet weight to dry weight was then calculated&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Histopathological examination</span><p id="par0060" class="elsevierStylePara elsevierViewall">The anterior lobe of the right lung &#40;tissue&#41; was placed in a 10&#37; formalin solution and processed routinely by embedding in paraffin and then tissues were sectioned into 5<span class="elsevierStyleHsp" style=""></span>&#956;m pieces and stained with hematoxylin &#38; eosin stain&#46; Pulmonary injury was graded into four grades as follows&#58; grade 0&#44; no diagnostic change&#59; grade 1&#44; mild neutrophil leukocyte infiltration and mild to moderate interstitial congestion&#59; grade 2&#44; moderate neutrophil leukocyte infiltration&#44; perivascular edema formation&#44; and partial destruction of pulmonary architecture&#59; and grade 3&#44; dense neutrophil leukocyte infiltration and complete destruction of pulmonary structure&#46;<a class="elsevierStyleCrossRef" href="#bib0315"><span class="elsevierStyleSup">19</span></a> A total of five slides from each lung sample were randomly screened&#44; and the mean was accepted as the representative value of the sample&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">The Statistical Package for the Social Sciences for Windows 11&#46;5 was used for statistical analysis&#46; All values were given as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; Differences between groups were statistically analyzed by Kruskal&#8211;Wallis test &#40;ANOVA&#41; and chi-square test&#46; Statistical significance was accepted as <span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Results</span><p id="par0070" class="elsevierStylePara elsevierViewall">All of the rats survived until the end of the study period&#46; Data belonging to histological changes from lung samples after reperfusion are represented in <a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#46; In the ischemia&#8211;reperfusion group&#44; tissues showed histological changes with partial destruction of pulmonary architecture&#44; neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46; Grades 1&#44; 2 and 3 of lesions were obtained in the ischemia&#8211;reperfusion group&#44; with an average of 2&#46;3&#46; These pathological changes in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; particularly neutrophilic infiltration and edema&#44; were much less than in the ischemia&#8211;reperfusion group &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Four animals in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group presented no injury and the others had lesions grades 1 and 2&#44; averaging 0&#46;7&#46; Histopathologically&#44; there was a significant difference &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; between the two groups&#46; The average of histological changes in the sham group &#40;0&#46;2&#41; was lower than the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; but with no statistical significance &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0075" class="elsevierStylePara elsevierViewall">Measurement of lung water content was used to estimate pulmonary edema&#46; Compared with the ischemia&#8211;reperfusion group&#44; the lung water content in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups was decreased significantly &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41;&#46; No significant difference in lung wet&#47;dry weight ratio was found between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">The NO level of lung tissue was increased in the ischemia&#8211;reperfusion group &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; The treatment with melatonin recovered this increase and caused a reduction in comparison with the ischemia&#8211;reperfusion group &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41;&#46; No significant difference in NO level was found between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">The novel indicator for neutrophil function&#44; MPO activity&#44; in the ischemia&#8211;reperfusion group was found to be increased dramatically compared to the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;000&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; MPO activity comparison between the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin and sham groups did not show significant results &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Discussion</span><p id="par0090" class="elsevierStylePara elsevierViewall">Peripheral arterial clamping&#44; as a surgical procedure&#44; is routinely used for orthopedic surgery or trauma in elective and emergency cases&#46; Lung damage has been shown to occur following transient arterial occlusion&#46; The ischemic damage resulted from a decrease in the blood flow to an organ&#46; When restoring blood flow a more pronounced damage&#44; so called reperfusion injury&#44; occurs&#46; In the development of ischemia&#8211;reperfusion injury&#44; the enhanced generation of oxygen radicals has been suggested&#46;<a class="elsevierStyleCrossRef" href="#bib0320"><span class="elsevierStyleSup">20</span></a> Free radicals have been previously demonstrated to play a significant role in the etiology of remote lung injury in animal models&#46;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">4&#44;21</span></a> It has been widely described that lung injury often occurs after skeletal muscle ischemia due to release of free radicals of reperfusing tissues&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">4</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Melatonin and its metabolites have been shown to have antioxidant properties in <span class="elsevierStyleItalic">in vitro</span> and <span class="elsevierStyleItalic">in vivo</span> systems&#44; both in cells and body fluids&#46;<a class="elsevierStyleCrossRefs" href="#bib0330"><span class="elsevierStyleSup">22&#8211;24</span></a> Furthermore&#44; melatonin plays an important role in activating antioxidant defences&#44;<a class="elsevierStyleCrossRefs" href="#bib0345"><span class="elsevierStyleSup">25&#44;26</span></a> and protects cells from oxidative loads and apoptosis induced by mitochondrial DNA deletion&#46;<a class="elsevierStyleCrossRef" href="#bib0355"><span class="elsevierStyleSup">27</span></a> Both effects allow melatonin to reduce the extent of reactive oxygen species&#44; improving the outcomes of oxidative related pathologies such as ischemia<a class="elsevierStyleCrossRef" href="#bib0395"><span class="elsevierStyleSup">35</span></a> and prevention of aging&#46;<a class="elsevierStyleCrossRefs" href="#bib0360"><span class="elsevierStyleSup">28&#8211;30</span></a>It has been demonstrated that melatonin can prevent ischemia&#8211;reperfusion injury in cerebral artery occlusion induced stroke&#44; in traumatic brain and spinal cord injuries&#44; in animal models&#46;<a class="elsevierStyleCrossRefs" href="#bib0375"><span class="elsevierStyleSup">31&#8211;33</span></a> Besides the generation of free radicals&#44; the ischemia&#8211;reperfusion injury is associated with exaggerated inflammatory response&#46;<a class="elsevierStyleCrossRef" href="#bib0390"><span class="elsevierStyleSup">34</span></a> Activated polymorphonuclear leukocytes aggregate and adhere to endothelium&#44; and impair blood flow and development of endothelial cell edema&#46;<a class="elsevierStyleCrossRef" href="#bib0395"><span class="elsevierStyleSup">35</span></a> Recently&#44; melatonin reduced morphological injury due to inhibition of neutrophils infiltration into the reperfused intestine with abolished P-selectin expression and intercellular adhesion molecule-1 up-regulation on endothelial cells&#46;<a class="elsevierStyleCrossRef" href="#bib0380"><span class="elsevierStyleSup">32</span></a></p><p id="par0100" class="elsevierStylePara elsevierViewall">Several methods have been used to define the role of neutrophils in reperfusion tissue injury&#46; MPO plays a fundamental role in oxidant production by neutrophils&#46; Neutrophils are a main potential source of oxygen-free radicals&#46;<a class="elsevierStyleCrossRef" href="#bib0400"><span class="elsevierStyleSup">36</span></a> A previous study clearly demonstrated that skeletal muscle ischemia&#8211;reperfusion led to remote lung inflammation&#44; which was characterized by increased MPO activity and significant neutrophil infiltration in the lung&#46;<a class="elsevierStyleCrossRef" href="#bib0405"><span class="elsevierStyleSup">37</span></a> NO level is a significant determinant of the lung injury process which is caused by lower extremity ischemia&#8211;reperfusion&#46;<a class="elsevierStyleCrossRef" href="#bib0410"><span class="elsevierStyleSup">38</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">The effects of melatonin on ischemia&#8211;reperfusion injury in an <span class="elsevierStyleItalic">ex vivo</span> rat heart model was studied by Lagneux et al&#46;<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">39</span></a> The authors explained that the melatonin significantly reduced infarct size and the incidence of reperfusion arrhythmias&#46;<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">39</span></a> In an experimental study Erkanl&#305; et al&#46;<a class="elsevierStyleCrossRef" href="#bib0420"><span class="elsevierStyleSup">40</span></a> reported that melatonin has a protective effect against skeletal muscle ischemia&#8211;reperfusion injury and can reduce the incidence of compartment syndrome after chronic or acute peripheral arterial occlusions&#46; Other studies have shown that melatonin protects kidney&#44;<a class="elsevierStyleCrossRef" href="#bib0425"><span class="elsevierStyleSup">41</span></a> liver&#44;<a class="elsevierStyleCrossRef" href="#bib0430"><span class="elsevierStyleSup">42</span></a> testis<a class="elsevierStyleCrossRef" href="#bib0435"><span class="elsevierStyleSup">43</span></a> and intestine<a class="elsevierStyleCrossRef" href="#bib0440"><span class="elsevierStyleSup">44</span></a> against ischemia&#8211;reperfusion injury&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">In the last few years&#44; Okutan et al&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">14</span></a> investigated the protective role of melatonin in lung remote organ injury&#46; For that purpose&#44; they induced lung injury by the occlusion of infrarenal abdominal aorta for 30<span class="elsevierStyleHsp" style=""></span>min and reperfusion for 12<span class="elsevierStyleHsp" style=""></span>h&#46; These authors administered melatonin intraperitoneally at 20<span class="elsevierStyleHsp" style=""></span>mg&#47;kg&#44; 1<span class="elsevierStyleHsp" style=""></span>h prior to trial&#46; They found that pre-treating rats with melatonin significantly decreases malondialdehyde &#40;MDA&#41; levels as well as MPO activity&#46; Based on this finding&#44; they believed that their study indicated the potential of attenuating lung injury&#44; and require further studies to show improvement in the morphology of the injured lungs&#46; In the current research&#44; we tested the hypothesis that 10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;v&#46; of melatonin could protect the lungs after 2<span class="elsevierStyleHsp" style=""></span>h femoral artery occlusion and 24<span class="elsevierStyleHsp" style=""></span>h reperfusion&#46; In this study melatonin inhibited MPO activity and decreased NO level which was increased by ischemia&#8211;reperfusion&#46; Pulmonary architecture&#44; neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema were much reduced in the treated group&#46; However&#44; there are further studies to be conducted to test the effects of various doses of melatonin treatment against ischemia&#8211;reperfusion injury in lung&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">Sharma et al&#46;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">18</span></a> showed that lungs undergoing ischemia&#8211;reperfusion displayed increased vascular permeability and pulmonary edema&#46; The conclusion of increased vascular permeability is an assumption based on the lung wet&#92;dry weight ratio&#46; Significant lung edema occurred after ischemia&#8211;reperfusion in rat which was attenuated by melatonin treatment&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">Data acquired demonstrates that melatonin can significantly decrease the severity of pulmonary edema&#44; decrease vascular permeability&#44; decrease leukocytes infiltration and inhibits cellular apoptosis&#44; after skeletal muscle ischemia&#8211;reperfusion&#46; Our findings support the potential use of melatonin as a therapeutic agent in the prevention of acute lung injury associated with skeletal muscle ischemia&#8211;reperfusion&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Ethical disclosures</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Protection of human and animal subjects</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association &#40;Declaration of Helsinki&#41;&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Confidentiality of data</span><p id="par0130" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Right to privacy and informed consent</span><p id="par0135" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Conflicts of interest</span><p id="par0140" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare&#46;</p></span></span>"
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          "titulo" => "Specimen collection"
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          "titulo" => "Biochemical assay"
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          "titulo" => "Lung wet&#47;dry weight ratio"
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          "titulo" => "Histopathological examination"
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              "titulo" => "Protection of human and animal subjects"
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    "fechaRecibido" => "2013-09-10"
    "fechaAceptado" => "2014-01-08"
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            0 => "Melatonin"
            1 => "Ischemia&#8211;reperfusion"
            2 => "Lung remote injury"
            3 => "Histopathology"
            4 => "Myeloperoxidase"
            5 => "Nitric oxide"
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    "resumen" => array:1 [
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Objective</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">This study evaluated the protective antioxidant effect of melatonin on lung injury as a remote organ after skeletal muscle ischemia&#8211;reperfusion in rats&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Thirty male Wistar rats were allocated randomly into three experimental groups&#58; operated with no ischemia &#40;Sham&#41; group&#44; ischemia&#8211;reperfusion group and ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#46; Hind limb ischemia was induced by clamping the femoral artery&#46; After 2<span class="elsevierStyleHsp" style=""></span>h ischemia&#44; the clamp was removed and the animal underwent 24<span class="elsevierStyleHsp" style=""></span>h reperfusion&#46; Rats in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group received melatonin &#40;10<span class="elsevierStyleHsp" style=""></span>mg&#47;kg i&#46;v&#46;&#41;&#44; immediately before the clamp was removed&#46; At the end of the trial&#44; animals were euthanized and the lungs were removed for water content determination&#44; histopathological and biochemical studies&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">In the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group&#44; tissues showed less intense histological abnormalities such as neutrophilic infiltration&#44; intra-alveolar hemorrhage and edema compared with the ischemia&#8211;reperfusion group&#46; Histopathologically&#44; there was a significant difference &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; between the two groups&#46; The lung water content in the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group was significantly lower than the ischemia&#8211;reperfusion group &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Lung tissue myeloperoxidase &#40;MPO&#41; activity and nitric oxide &#40;NO&#41; level were significantly &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; increased by ischemia&#8211;reperfusion&#46; The increase in these parameters was reduced by melatonin&#46;</p><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Comparing the ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin group with the sham group&#44; no significant increase in all analyzed aspects of the research was observed&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusions</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">These findings suggest that melatonin has preventive effects in lung tissue injury after transient femoral artery occlusion&#46;</p></span>"
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                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Groups&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">MPO &#40;U&#47;g protein&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">NO &#40;&#956;mol&#47;g protein&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top">Sham&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;47<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;11<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">&#42;</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">58&#46;07<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&#46;45<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">&#42;</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">8&#46;12<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;37<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">&#42;</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="table-entry  " align="left" valign="top">Ischemia&#8211;reperfusion<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>melatonin&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;7<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;17<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">&#43;</span></a>&nbsp;\t\t\t\t\t\t\n
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                    0 => array:2 [
                      "titulo" => "The pathophysiology of skeletal muscle ischemia and the reperfusion syndrome&#58; a review"
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ISSN: 21735115
Original language: English
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