The Experimental Study Of Controlled Reperfusion After Severe Limb Ischemia

Material and Methods:ⅠEstablish the in vivo model of controlled reperfusion after hind limb ischemia of rat.Acute hind-limb ischemia was first induced in Wistar rats.Unlike previous tourniquets models,femoral vessels were surgically prepared to facilitate controlled reperfusion and to prevent venous stasis.Then Controlled selective reperfusion is achieved with a microcatheter and the superficial epigastric artery as conduit.The femoral vein is clamped,and sequestered blood is washed out through a venous incision.ⅡThe local and system effect of Controlled reperfusion after severe hind limb ischemia.Rats were randomized into an experimental group(n=7),in which limbs were selectively perfused with a cooled isotone heparin solution at a limited flow rate before blood flow was restored,and a conventional group(n=7;uncontrolled blood reperfusion).Rats were killed 4 hours after blood reperfusion.Nonischemic limbs served as controls.ⅢThe mechanism of the protective effect of controlled reperfusion for ischemic hind limb limb of rat.Rats were randomly divided into sham group(n=5),traditional ischemia-reperfusion group(n=10),controlled reperfusion group(n=10),Fetpps intervention group(10),with the exception of sham group,the other three groups were randomly divided into two subgroups(reperfusion 0h,4h) respectively,each subgroup includes five rats.After reperfusion 0,4hour,the rats were killed.The samples were collected for detection of skeletal muscle content of MDA,plasma CK,muscle nitrotyrosine formation levels;and the related protein levels of P13 K / Akt signal transduction pathway activation were detected by Western blot.Results:1.Tecnincally,controlled reperfusion for the ischemic hind limb of rat is feasible with a microcatheter and the superficial epigastric artery as conduit.,this model is practical and reliable.2.Ischemia/reperfusion injury was significant in both groups;total wet-to-dry ratio was 159±44%of normal(P < 0.05 ),whereas muscle viability and contraction force were reduced to 65±13%(P<0.05 ) and 45±34%(P<0.05 ),respectively. Controlled reperfusion,however,attenuated reperfusion injury significantly.Tissue edema was less pronounced(132±16%versus 185±42%;P<0.05) and muscle viability(74±11%versus 57±9%;P<0.05) and contraction force(68±40%versus 26±7%;P<0.05) were better preserved than after uncontrolled reperfusion. Moreover,subsequent blood circulation as assessed by laser Doppler recovered completely after controlled reperfusion but stayed durably impaired after uncontrolled reperfusion(P<0.05).3.The plasma CK,and tissue MDA level were decreased compared with uncontrolled blood reperfusion(P<0.05).At the protein level,The formation of the putative peroxynitrite footprint 3-nitrotyrosine was decreased after controlled reperfusion(P<0.05),and the PI3-kinase/Akt signaling pathway was activated with the increased level of phosphation of Akt level(P<0.05). Conclusions:1.The first time to establish a reliable,practical in vivo model to test controlled reperfusion after severe hind limb ischemia of rat.2.Reperfusion injury was significantly alleviated by basic modifications of the initial reperfusion period in a new in vivo model of acute limb ischemia.3.The protective effects of controlled reperfusion in the rat ischemic limb is through regulating PI3 K-Akt signal pathway.Perhaps,its mechanism may be related to reduction of free radical injury and strengthen the anti-oxidation effect.4.With this model,systematic optimizations of according protocols may eventually translate into improved clinical management of acute limb ischemia.Prospect:These findings highlight the enormous clinical potential of controlled reperfusion and,as a benchmark for refinements,provide the methodological framework for systematic research into an optimized and clinically applicable reperfusion protocol that may eventually translate into improved patient care.