| Ischemia of the extremities is an unavoidable occurrence during peripheral vascular injury, aortic aneurysm surgery, replantation of extremities. Although full reperfusion of ischemiac limbs is necessary during resuscitation, there is strong clinical and experimental evidence that reperfusion of ischemic tissue might be associated with remote organ injury, such as heart, lung, liver, small intestine and etc. The mechanisms of acute injury following extremity ischemia-reperfusion (I/R) are unknown at present. It has been found that the adhesion, infiltration and inflammatory factor from polymorphonuclear neutrophil (PMN) is the key point during the process of I/R injury, which is controlled by the activation of nuclear transcription factor-kappa B (NF-kB). The gene expression of ICAM-1 is the premise for adhesion and aggregation of PMN, which is controlled by NF-kB. However, there is little report about the role of NF-kB in hind limb I/R injury.In order to determine the effect that limb I/R could induce the activation of NF-kB/ I-kB system and related genes in skeletal muscle and remote organs. We investigated the role of NF-kB by administering PDTC, an inhibitor of NF-kB, and NF-kB decoy ODNs, a double chain ODNs, which could combine to the gene site of NF-kB and inhibit the activation of related genes, in the skeletal muscle and remote organ injury following hind limb I/R. The aim of this study was to interpret the complicated pathophysiological process.MethodsAnimal model and Experimental protocolsMale Wistar rats (250g+50g) were anesthetized with intraperitoneal administration of sodium pentobarbital (40mg/kg) and secured in a supine position. Common femoral arteries were clamped in the left groins with a non-injury tourniquets wrapped around thigh at the proximal of the leg. Removal of the clip allowed for reperfusion for 1 hours, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours after ischemia for 4 hours. 54 rats were randomly divided into 3 groups: sham operation group (n=6), ischemia group (n=6), I/R group (n=42). The I/R group received 4 hours of ischemia and then were given from 1 h to 48h of reperfusion. The blood sample, skeletal muscle, both lungs and kidneys were harvested at the end of the experiment. On the other hand, PDTC group(n=12), NF-kB decoy or scamlbe decoy group(n=12, each group) were also established by perfusion of PDTC(100mg/kg, dissolved in saline) and decoy(200ug)by femoral artery injection prior to reperfusion and samples were collected 8 hours and 24 hours after reperfusion. Histological assessmentAfter harvest, representative blocks from the resected skeletal muscles, lungs and kidneys were processed through formalin fixation and routine paraffin embedding. Sections were stained with hematoxylin and eosin. Lung PMN sequestration was quantitated by counting alveolar septal wall PMN. Lung wet-to-dry weights ratio (W/D)Parenchymal samples of whole lung tissue were blotted and weighed. Tissues were weighed again another 24 h later to verify complete dehydration. Data were calculated as wet weight divided by dry weight and used as an indicator of pulmonary edema. Measurement of lung tissue MDA and MPOAcute lung or kidney injury was determined with measuring the contentof malondialdehyde (MDA) and myeloperoxidase (MPO) as a marker of lipid peroxidation. The MDA and MPO contents in the supematants were measured using MDA or MPO assay kit from Nanjing Jiancheng Corp (China). Reverse transcription-polymerase chain reaction (RT-PCR)Total RNA was isolated from lung tissue with Trizol reagent, then mRNA was reverse transcribed to generate cDNA using standard methodology. The experimental conditions for PCR: 94℃denaturation for 2 min, then 30 cycles for 30 seconds at 94℃, 30seconds at 55℃, and 45 seconds at 72 ℃, extention at 72 ℃ for 5 min at last. After amplification, 5uL of PCR product per lane was resolved on 2% agarose gel containing ethidium bromide. Bands were confirmed under UV fluorescence. A(gene expression)=NF-KB p65(ICAM-l, I-KB|3)/p...
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