Effects Of Noninvasive Limb Preconditioning On Myocardial Ischemia-reperfusion Injury In Rats

IntroductionIschemic preconditioning (IPC) refers to a mechanism whereby brief periods of ischemia - reperfusion (IR) render a tissue relatively resistant to the harmful effects of a subsequent prolonged period of ischemia. This phenomenon was first described by Murry who showed that IPC decreased infarct size induced with 40 minutes of coronary artery occlusion in a canine model of myocardial ischemia. Ischemic preconditioning is one of the most effective methods against ischemia reperfusion injury. The original experimental models of classical ischemic myocardial preconditioning involved brief complete obstruction of the infarct - related coronary artery before a long episode of ischemia. Despite its potent infarct - protective effect, the technique of local IPC may not be acceptable to most surgeons because it requires a prolonged operative time, and more importantly , there is always the risk of damaging the vessel due to repeated clamping. Although animal studies have shown that protection occurs locally in the tissue being preconditioned, systemic effects of localized IPC have been observed. This phenomenon was termed " intraorgan" remote IPC. Remote myocardial protection has been shown with preconditioning kidney, mesentery, and even skeletal muscle. Study demonstrated that a brief cycle of lower - limb ischemia -reperfusion protected the heart against tachyarrhythmia in rats. The mechanisms by which remote preconditioning protects the heart are less well understood. The purpose of this study was to assess the effect on myocardial infart size of nonin-vasive hindlimb preconditioning by tourniquet application, and to determine whether nitric oxide ( NO) , neurogenic pathway and NF - κB play an important role in the mechanism of acute remote IPC.Materials1. Experimental animals: 96 Wistar rats, regardless of sex, provides by Experimental Animal Center, China Medical University.2. Experimental instruments: Ohmeda 7000 Aneshesia machine ( Ohmeda, U. S. A. ) , Animal ventilator ( Havard 638, U. S. A. ) , Olympus light microscope (Japan) , PTC - 100 PCR amplification device ( U. S. A. ) , Dupont ST -21 low temperature and high speed centrifuger( U. S. A) , KADAK ID gel image analyzing system ( USA) , GIS - 700 D digitized gel scanning analyzing system (Shanghai).3. Chemical and reagents: pentobarbital sodium (ShangHai) , N[G] - Nitro - L - Arginine Methyl Ester ( L - NAME ) , hexamethonium chloride, and ProDTC were obtained from the Sigma Chemical Co.MethodsEffect of noninvasive limb preconditioning on myocardial ischemia - reper-fusion injury. 32 Wistar rats were randomly allocated to one of the four experimental groups, C Group: control group, the rats underwent thoracotomy only; I Group: ischemia/reperfusion group, the rats undergwent 30 min occlusion of the left anterior descending coronary artery, and 120 min reperfusion; PC Group: classic myocardial preconditioning, the rats underwent four cycles of 5 min occlusion and reperfusion of left anterior descending coronary artery before the experiment was continued as I Group; PL Group: noninvasive limb ischemic preconditioning group, the rats underwent four cycles of 5 min occlusion and reperfusion of both hindlimbs using a tourniquet before the experiment was continued as in Pc Group, limb ischemia was produced by placing a thin elastic tourniquet (1 - cm diameter) around the upper third of the hind extremities. Hemodynam-ics were measured throughout the experiment. At the end of experiment, 2 ml blood was withdrawn for the measurement of LDH and CK. The left anterior descending coronary artery was occluded again, and the area at risk (AAR) and infarct size (IS) were determined by TTC staining.-) ? The mechanism of noninvasive limb preconditioning on myocardial ischemia - reperfusion injury. 80 Wistar were randomly allocated to one of the five experimental groups. I Group: ischemia/reperfusion group; PL Group: noninvasive limb ischemic preconditioning group; PL_NGroup: L-NAME + limb preconditioning group, received 10 mg/kg L-NAME IV. before limb preconditioning, then the experiment was continued as in PL Group; PL_HGroup: hexamethonium chloride + limb preconditioning group, received hexamethonium chloride 20 mg/kg IV before limb preconditioning, then the experiment was continued as in PL Group; PL_DGroup: proDTC + limb preconditioning group, received peritoneal injection of proDTC 125 mg/kg before limb preconditioning, then the experiment was continued as in PL Group. At the end of experiment, the AAR and IS were determined by TTC staining. And another 8 rats in each group, all rats were killed and myocardium were stored in liquid nitrogen for the measurement of SOD, MDA, NO, NOS, iNOS, iNOS m RNA and NF - kB m RNA.Results1. There were no significant differences in MAP and HR among these groups at any time point during the experiments (P >0.05).2. The level of LDH and CK in I Group were significantly increased compared with C Group ( P <0. 05). There were no differences among Pc Group, PL Group and C Group.3. There were no significant differences of AAR among all the experimental groups. The myocardial infarct size ( IS) was decreased significantly in Pc Group, PL Group, PL_HGroup compared with I Group (P <0. 05). And there were no significant differences in PL_NGroup, PL_DGroup compared with I Group (p>0.05).4. In PL and PL_HGroup, SOD increased and MDA decreased comparedwith I Group. There were no differences between I Group and PL_NGroup.5. The level of NO, NOS, iNOS, iNOS mRNA were increased in PL and PL_HGroup compared with I group ( P <0. 05) , and no significant changes in PL_N Group.The NF - kB mRNA is weaker in PLGroup than that in I Group, but is stronger than that in PL_DGroup (P <0.05). There was almost no expression of NF - kB mRNA in PL_DGroup.DiscussionIschemic preconditioning (IPC) refers to a mechanism whereby brief periods of ischemia - reperfusion (IR) render a tissue relatively resistant to the harmful effects of a subsequent prolonged period of ischemia, and result in myocardial protection. Ischemic preconditioning is one of the most effective methods against ischemia reperfusion injury. Many studies have demonstrated that IPC not only happened to the tissue being preconditioned, but also have systemic effects. Remote myocardial protection has been shown with preconditioning myocardial, kidney, skeletal muscle, and even intestinal tissue. Study reported that a brief cycle of lower - limb ischemia - reperfusion protected the heart against tachyarrhythmia in rats. But the mechanisms by which remote preconditioning protects the heart are less well understood.Our study was to assess the effects noninvasive limb preconditioning on ischemic reperfusion myocardium. The results shown that a noninvasive remote IPC technique was just as effective as the classic IPC on protecting myocardium against infarction. We further demonstrated that this infarct - protective effect of the remote IPC was abolished by intravenous pretreatment with a nonselective NO synthase antagonist, L - NAME, and specific NF - kB antagonist, ProDTC. Ganglionic blocker hexamethonium chloride, did not affect the infarct - protective effect of this remote IPC. NO and NF - kB play an important role in the mechanism of this acute remote ischemic preconditioning and the neurogenic pathway seems not involved in this acute noninvasive limb preconditioning.Unlike previous animal models, the remote IPC techniques discussed here...