| Objective:TO investigate the effects of 3 different protocols of limb ischemic preconditioning to ischemia reperfusion injury of skeletal muscle in rats, through the light microscope and the ultrastructure of skeletal muscle were observed in skeletal muscle, determination of the wet/dry ratio, serum lactate dehydrogenase (LDH) and creatine kinase (CK) activity, detection of skeletal muscle necrosis, differences in the protective effect comparison of 3 different ischemic preconditioning protocols on ischemia reperfusion injury of limbs, to select the best solution from it, which can lay the foundation for further experimental research.Methods:The use of noninvasive arterial occlusion in rat right hindlimb femoral artery reperfusion, loosen the artery clamp, establish a model of limb ischemia-reperfusion injury. Healthy adult male SD 48 rats, were randomly divided into ischemia reperfusion group (ischemia group), ischemia preconditioning, A B, C group 4 group (n=12/group), ischemia reperfusion group in ischemia reperfusion group, the occlusion of the femoral artery after 4 hours of reperfusion. Ischemia preconditioning group A by adjustment of occlusion of the femoral artery for 2 minutes, open the folder for 2 minutes, and 6 cycles, then the occlusion of the femoral artery ischemia 4 hours after reperfusion. Ischemia preconditioning group B by adjustment of occlusion of the femoral artery for 5 minutes, open the folder for 5 minutes, and 3 cycles, then the occlusion of the femoral artery ischemia 4 hours after reperfusion. Ischemia preconditioning group C by adjustment of occlusion of the femoral artery for 1 minutes, open the folder for 1 minutes, and 10 cycles,4 hours after ischemia, reperfusion. Ischemia group and ischemia preconditioning group were reperfusion after 2 hours,12 hours,24 hour time point of femoral venous blood and left, right hind leg muscle, and the rats were killed. The femoral vein blood serum were measured by centrifugation, LDH and CK activity by ELISA method. Take the right hindlimb soleus muscle, changes with H & E staining pathological observation of inflammation, edema and necrosis of skeletal muscle, stained with uranyl acetate and lead citrate, change the transmission electron microscope was used to observe the ultrastructure of skeletal muscle. Take the left (blank control), right hindlimb gastrocnemius weighed, dried and weighed dry weight, wet to dry weight ratio calculation. Cut the right hindlimb tibialis anterior muscle, using TTC staining, observe the color changes of muscle necrosis area and calculate the percentage of using image software, the experimental results were compared.Result:Ischemia reperfusion group at three time points (2 hours,12 hours, 24 hours) of the CK and LDH values were higher than the preconditioning group (P<0.05); 2 hours after reperfusion, the preconditioning group A CK value and LDH value is lower than B, C group (P<0.05), pre the adjustment of group B CK and LDH values lower than C group (P<0.05); 12 hours after reperfusion, the preconditioning group A CK and LDH values had no significant difference with B group (P>0.05), preconditioning group A CK and LDH values were lower than those in group C, reperfusion 24 hours, and LDH were lower than A, C group B group CK preconditioning values (P<0.05), the preconditioning group A CK value and LDH value is lower than the C group (P <0.05). The wet/dry weight ratio:ischemia reperfusion group after 24 hours of wet and dry weight ratio, the preconditioning group A after reperfusion 2 hours minimum ratio, the ratio of ischemia group at different time points were higher than the preconditioning group (P<0.05), the ratio of reperfusion within 12 hours of the preconditioning group A were less than B, group C (P<0.05), the ratio of the preconditioning group B than group C (P<0.05); 12-24 hours after reperfusion, the ratio of the preconditioning group B is lower than A, C group (P <0.05), the ratio is better than that of C group A preconditioning group (P< 0.05). The results of TTC staining:the ischemia necrosis area were higher than the preconditioning group three,12-24 hours after reperfusion, the preconditioning group B necrotic area at least. HE staining showed ischemia reperfusion for 24 hours showed myofibrillar degeneration twisted edema, stripes and structural disorder, part of the regional nuclear pyknosis disappeared, vascular congestion, neutrophil infiltration in the preconditioning group A, preconditioning group B, preconditioning group C 24 hours of injury of skeletal muscle were lighter than ischemia group, the preadjustment of A group, the preconditioning group B skeletal muscle injury at least. Electron microscopy showed ischemia reperfusion for 24 hours were severe edema, disorganized, myofilament dissolved fracture, reduced glycogen granules, mitochondria swelling, sarcoplasmic reticulum dilatation.24 hours after reperfusion, the preconditioning group A, preconditioning group B, preconditioning group C disorder of myofibrils and horizontal structural damage, swelling of mitochondria and sarcoplasmic reticulum expanded Zhang Junming was lighter than the ischemia group, the preconditioning group B ultrastructure at least. Conclusion:ischemia preconditioning can reduce limb ischemia reperfusion injury by decreasing CK and LDH contents in plasma, reduce skeletal muscle necrosis area, reduce the degree of injury, which play a protective role. The protective effect of three kinds of ischemia preconditioning scheme has the difference, and protect the ischemia preconditioning with time limit. Comprehensive analysis, in the preadjustment scheme to protect effect of 3 cycles of 5 min ischemia reperfusion of the optimal/5 minutes.Conclusion:Ischemia preconditioning can reduce limb ischemia reperfusion injury, by reducing CK, LDH content in plasma, reduce skeletal muscle necrosis area, reduce the degree of injury, which play a protective role. The protective effect of three kinds of ischemia preconditioning scheme has the difference, and protect the ischemic preconditioning with time limit. Comprehensive analysis, in the ischemic preconditioning protocols, the best one is 3 cycles of 5 min ischemia reperfusion/5 minutes reperfusion protocol. |
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