| Limb replantation is an important clinical practice in the field of hand surgery. The development of microsurgical techniques and scientific technologies have led to greater success in replantation, therefore, shifting clinicians'focus from survival rate to improving postoperative limb condition. One of the challenges is to reduce replanted tissue damages, predominantly the skeletal muscle and nerve tissue damages, which often follow the operation. Reperfusion injury, one of the complications that occur after replantation, causes severe damages to extremity function. How to alleviate such injury, therefore, is of vital importance to the restoration of extremity function. Currently, clinicians in hand surgery field are seeking diligently a feasible method to reduce extremity ischemia and reperfusion injury post limb replantation, in hopes of restoring the function of extremity to a better degree. Over the years, medical researchers home and abroad have put in efforts to study the mechanisms of, and look for ways to prevent or cure reperfusion injury to skeletal muscles. The progress made in basic research, however, has not led to significant breakthrough in clinical practice. In this study, we improved a previously established animal reperfusion model. Directing against various key events that occur during ischemia reperfusion injury, we combined several common clinical drugs and developed a perfusate. We filled the animal limb with the perfusate before replantation, and tested the protective effects of the perfusate against ischemia reperfusion injury. The animal limb reperfusion model that we used was established with the hindlimb of rat. The perfusate contained decacortin, vitaminC, verapamil, and A.C.I composita pro injectione. Before replantation, we filled the limb with perfusate and injected sodium aescinate into the body through the great saphenous vein. We then performed replantation to rats. At each of various time points, i.e. 1, 2, 4 or 8 hours post replantion, a group of rats was sacrificed, various tests were performed and results were analyzed with statistical method. In control group, rats received replantation without pretreatment of perfusate or sodium aescinate and then were sacrificed at corresponding time points as were the experiment groups. 1. The water level in tibialis anterior muscle: In control group, with time after re-filling, the water content of tibialis anterior muscle increased continuously, especially in the first hour. The experimental group showed steady water increase in the first eight hours after re-filling and level of water content was significantly lower as compared to the control group. The perfusate can relieve the water content of tibialis anterior muscle evidently. 2. The MDA level in skeletal muscle: The MDA level in skeletalmuscle was much lower in the experimental group than that in the control group. The MDA level changed significantly in the first two hours after re-filling in the experimental group. 3. The superoxide dismutase (SOD) activity in skeletal muscle: The activity of SOD reflects the ability of the tissues to clear free radical. After treated with persuate , the activity of SOD in limb was much higher in experimental group than that in control group, especially in the first hour . So we can conclude that: If the limb is perfused with perfusate before replantation, the activity of SOD will increase in a short time after reperfusion . This can reinforce the tissue ability against reperfusion injury by free radical. 4. The mitochondrion ATP enzyme activity in skeletal muscle: The activity of mitochondrion ATP enzyme increased significantly in experimental group that was pretreated with perfusate than that in the control group. In the initial four hours, the activity of mitochondrion ATP enzyme varied evidently with time. But from four to eight hours, this tendency of change gradually diminished, and the reperfusion injury began to decrease. 5. Nitrogen monoxide level in plasma: In control group, the nitrogen monoxide level plasma increased slightly in the first hour and then decreased greatly with time. But in the experimental group, the nitrogen monoxide level did not increase after reperfusion. It showed gradual and steady decrease with time. The reperfusion injury caused by NO in experimental group is less severe than in control group. 6. â…§factor correlated antigen level in blood vessel endothelial cells: mRNA level of the antigen was measured using RT-PCR. Theâ…§factor correlated antigen mRNA level increased gradually with time in control group in the initial four hours after reperfusion, and began decrease after four hours, and finally dropped to the initial level by eight hours . In experimental group: the trend of mRNA change with time was the same as was seen in control group, but the mRNA level was much lower than that in control group. 7. The TM level in blood vessel endothelial cells: TM mRNA level was measured using RT-PCR. Experiment group showed gradual increase in TM mRNA level within four hours post reperfusion, and decrease from four to eight hours. At eight hours, the level of TM mRNA dropped to basal level. There was no significant mRNA level change in control group. After filling with perfusate, TM transcription level was up-regulated. TM has anti-coagulation effect. Therefore, up-regulation of TM transcription can reduce the level of thrombosis after replantation. 8. The myeloperoxidase (MPO) level in skeletal muscle: In control group, the level of MPO increased significantly in the initial four hours post reperfusion, but continued to increase at a much lesser degree after four hours. The reperfusion injury decrease with time accordingly. The result in the experimental group is better than that in control group. 9. The surface expression level of CD11a / CD18 on neutrophilic granulocytes: The expression of CD11a / CD18 increased with time after reperfusion in both groups, especially in the initial two hours. But the expression level in experimental group is much lower than that in control group. 10. The expression level of ICAM-1 in skeletal muscle bloodvessel: The expression of ICAM-1 increased after reperfusion, and the changes were significant from two to four hours. The level of ICAM-1 was affected by the reperfusion injury significantly during the same period. In the experimental group, the changes were significant in the first hour. This time difference was distinct between the two groups. 11. The iNOS level on skeletal muscle blood vessel: After reperfusion, the level of iNOS began to increase, and the positive rate (number of vessels containing high level of iNOS) is high in control group. Four hours later, the iNOS level began to decrease. In the experimental group, the positive rate was much lower after reperfusion than the control group. The perfusate can decrease the level of iNOS , but can't alter the trend of level changes with time . 12. Electron microscopy: The myofilament lined up in order in the normal skeletal muscle, the mitochondria were eumorphism . In control group : 0.5 hour after reperfusion , the structure of muscle fiber showed disorder and edema , the mitochondria were swelling and dilating , the mitochondrial ridge became blurry and partial damages were observed ; 4 hours later , Local myofilament was disrupted , mitochondria decreased and swelled ; 8 hours later , myofilament was disrupted more severely , sarcomere disappeared , sarcoplasm increased significantly , the mitochondrial ridge shapes were vacuoles , pyknosis was seen in blood capillary endothelial cell , chromatin agglutination , there were lots of pinocytosis vesicles in cytoplasm . In experimental group : 0.5 hour after reperfusion , myofilament clouding , partial mitochondria swelling , stroma hyperplasia ; 4 hours later , sarcomere clear , mitochondria swelling , mitochondrial ridge disrupting ; 8 hours later , sarcomere clear ,mitochondria swelling , mitochondrial ridge disrupting , there were many mitochondria , blood capillary endothelial cell chromatin tendency to border, erythrocyte and Granulocytic cell were seen in Lumen of blood vessel , the Lamina basalis thickness were different on the two sides . From the results, we have found that the reperfusion injury remained severe within 4 hours after reperfusion. After 4 hours, however, the injury level began to decrease, and the effect of various metabolites began to drop as well. The perfusate, as observed, can significantly reduce the reperfusion injury. Filling the limb with perfrusate before replantation, therefore, can protect the tissues of extremity, especially the skeletal muscle, against reperfusion injury, and ultimately restore the function of replanted extremity to a better degree. In this study, we have improved the animal model of reperfusion injury and rendered a better reflection of true clinical condition. The experimental results obtained from this model, therefore, have reflected more closely the real limb replantation. The subsequent conclusions generated from such results, therefore, are more realistic and better suited for clinical needs. At the same time, the method we took to dispense the perfusate into body -through the branch of femoral artery and the great saphenous vein -successfully avoids the harm to the important tissues of the limb and can be put into clinical use. Therefore, the model we generated here and the method of perfusion we applied are more realistic, practical and feasible than previous models. To truly reflect the mechanisms of reperfusion injury in reality,we chose the experimental index and the detecting methods generally accepted by previous ischemical reperfusion injury studies. The levels of water in tibialis anterior muscle, MDA in skeletal muscle, and myeloperoxidase in skeletal muscles reflect the severity of tissue damage caused by reperfusion injury. The testing methods are simple and convenient. The activity of superoxide dismutase reflects the tissues'ability to counteract the reperfusion injury. The mitochondrion ATP enzyme activity explains that the ischemical reperfusion injury has a close relationship with energy metabolism. The effect of Nitrogen monoxide in plasma is not yet clear, possibly both protective and damaging to a certain degree. The expression level changes of factor â…§correlated antigen , TM , ICAM-1 and the iNOS tells us that these factors can cause damage to blood vessel and microvessel and contribute to the "no reflow"Phenomenon. The result of electron microscopy is relatively direct and reliable, and not easily affected by unrelated experimental conditions. The expression of CD11a / CD18 on neutrophilic granulocyte surface reflects the condition of the lymphocyte and explains the role of inflammatory cell in reperfusion injury. Therefore, the reperfusion injury is affected by many factors that are individually important as well as interrelated. As a result, to prevent and cure of reperfusion injury, we must consider all possibly involved factors when developing a better treatment, in hopes of reducing the injury to the lowest degree.
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