Experimental Research On Perfusing Preservation And Replantation Of Rat Amputated Limb

Objective:To decrease ischemic and anoxia injury in amputated limbs, keep the tissue activity, and prolong the time limit of tissue degeneration and replantation. Extend the preservative time of amputated limbs. Reduce the rate of disability and promote functional recovery of amputated limbs and a better life condition after replantation.Methods:90 adult and healthy Wistar rats, weighted 250-300g, were used in the experiment which is separated into 4 parts. Part Ⅰ Experimental comparison of the preservated effects on different perfusates with single-shot perfusion of rat amputated hindlimbs:40 Wistar rats were randomly divided into 4 groups (10 rats in each group) for providing the models of amputated limbs. The first group was perfused with saline for 1h after amputation.The first group was perfused with saline.The second group was perfused with HTK solution. The third group was perfused with UW solution.The forth group, as the control group, was not perfused with any solution. All of amputated limbs were preserved in the refrigerator at 4 ℃, and the first three groups were perfused at the speed of 12ml/h for 1h just after the amputation. Samples of muscle tissue were taken for pathological (HE, PTAH and IHC-P) and biochemical examination every 6h, and ultrupathological examination every 12h.Part Ⅱ Experimental comparison of the preservated effects on different perfusates with every-12h-perfusion of rat amputated limbs:30 Wistar rats were randomly seperated into 3 groups (10 rats in each group) for providing the models of amputated limbs. The first group was perfused with salin.The second group was perfused with HTK solution. The third group was perfused with UW solution.All of groups were preserved in the refrigerator at 4 ℃, and were perfused at the speed of 12ml/h for 1h every 12h. Samples of muscle tissue were taken for pathological and biochemical examination every 6 hours and for ultrupathological examination every 12h. PartⅢ Experimental comparison of the preservated effects of rat amputated limbs on different perfusing methods with the UW solution:Compare the results of the UW-solution-perfusion-group in Part Ⅰ and Part Ⅱ. ParⅣExperimental comparison of the rat amputated limbs replantation after UW solution perfusionand 4℃ perservation: 20 Wistar rats were randomly divided into 2 groups, experimental group and control group (10 rats in each group). The two groups both harvest the right hindlimbs with amputation. Experimental group was perfused with 10ml UW solution through the inferior epigastric artery. Control group were perfused with nothing. Both groups were preserved in the refrigerator at 4℃ for different period amd replantated. Survival rate of both groups after surgery were recorded, and pathological examination (HE) of muscle tissue was taken.Results:Part I:during the cryopreservation, the index of ALP、 ALT、AST、CK、LDH、 LAC of perfusion group are lower than the non-perfusion group, while GLU and SOD of perfusion group are higher than non-prefusion group. The indexes between the UW-solution-perfusion group and non-perfusion group have stastical difference, while parts of the indexes among the other groups have. The pathological change of muscle tissue were lighter in perfusion groups than non-prefusion group.Among the three perfusion groups, the UW-solution-perfusion had the lightest pathological change. The percentage area of cell has statistical difference, non-perfusion group has the smallest; before 12h, saline-perfusion group has the largest, while after 12h UW-perfusion group gets to the top. IHC-P has statistical difference among the four groups at 24h. Part II: during the cryopreservation, parts of the biochemical indexes have statistical difference with others, UW-solution-perfusion group has lower indexes of ALP、ALT、AST、LDH、 SOD than saline-perfusion group.The pathological change of muscle tissue between UW-solution-perfusion group and HTK-solutin-perfusion group are similar, but lighter than those in saline-perfusion group.The result of percentage area of cell has statistical difference, saline-perfusion group has the largest cell area ratio. IHC-P IOD has no statistical difference among the three groups. PartⅢ:Single-shot UW solution perfusion group has no statistical difference in biochemical indexes, percentage area of cell and 1HC-P IOD with 12h-interval UW solution perfusion group. And the two groups have similar pathological and ultrapathological changes. ParⅣ:60% rats survived in experimental group, and 30% survived in control group. HE stain shows that skeletal muscle tissue in experimental group had lighter ischeamia-reperfusion injury than those in control group.Conclusion:1、Amputated limbs treated with perfusates and cryogpreservation had stronger protective effects than those treated with only cryogpreservation.2、Perfusing once with saline,HTK solution and UW solution, UW solution had strongest protective effects on amputated limbs.3、Perfusing every 12h with saline,HTK solution and UW solution, UW solution had strongest protective effects on amputated limbs.4、It had the same protective effects on amputated limbs treated by perfusing once and perfusing every 12h preserved for 24h.5、Cold ischeamia time in 3-5h, amputated hindlimbs perfusing with UW solution once and preserving at 4℃ had higher survival rate than those only preserved at 4℃ after replantation. And limbs had lighter pathological injury postsurgery.