Study On The Protective Effects Of Cold Plasma Cardioplegia On Myocardial Ischenia Reperfusion Injury In Infant Rabbit

PrefacePersistent myocardial ischemia is known to result in cell injury and death. Reperfusion is very important for myocardium,but attachment of leukocyte-en-dothelial cell,aggregation of platelet-leukocyte and transendothelial leukocyte migration can result in cell death after reperfusion of ischemia myocardium,in addition to activation of complement and production of active oxygen free radi-cals also leads to severer myocardial damage,namely myocardial ischemia reperfusion injury.During the open heart operation,the coronary flow is blocked,which leads myocardium to be ischemic and hypoxemic.By the end of the operation,the coronary flow is recovered which leads to new myocardial in-jury,namely reperfusion injury.It is a key task in the field of cardiac surgery how to reduce myocardial ischemia reperfusion injury.The modified hyperpotassium cardioplegia was developed since Melrose cre-ated the hyperpotassic cardioplegia in 1955,which improved the effect of myo-cardial protection and decreased operative mortality.It is out of place or lacks sound academic and experimental basis to protect the immature myocardium u-sing the cardioplegia suitable to mature myocardium.Recently the research on immature myocardial protection is being as a hotspot in myocardial protection.In this study,to investigate the protective mechanism of cold plasma cardioplegia on immature myocardium,we measured 1.creatine phosphokinase co-enzyme (CK-MB),lactec dehydrogenase co-enzyme(LD₁/LD₂)and adenosine triphosphate(ATP)content in coronary outflow,2.malonaldehyde(MDA), superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)content in myocardium,3.myocardial tumor necrosis factor(TNF)mRNA and protein, Bcl-2 mRNA and cardiomyocyte apoptosis.Animal;Thirty-two healthy infant rabbits(age 14-26 days)weighting 350-580g were used in this study.Four were as control,and other 28 were di-vided averagely into two groups-plasma group(P group)and crystalloid group (C group).Major instruments;constant temperature pump,comminuter,LKB-V ul-tramicrotome,HERMLEZ383K hypothermic high-speed centrifuger,KODAK ID gel-imaging system analysoa and PTC-100PCR extender.Major reagents;Bcl-2 Takara PCR reagent,MDA,SOD,GSH-PX a-gent kit etc.TNF immunohistochemistry kit,TNF in situ hybridization detecting kit.Methods1.Preparation for the isolated heart and Collection of samples;.After anes-thesia by injection of 846 0.3 ml/kg intramuscularly and heparinization by hepa-rin-sodium 400 u/kg intravenously,the infant rabbits heart were excised rapid-ly,rinsed thoroughly in chilled 4℃saline and then suspended on a cannula, and the perfused retrogradely in the isolated Langendroff model with 37℃oxy-genated K-H buffer solution at a constant pressure of 60 cmH₂O for 10 minutes and 10 minutes coronary outflow solution was collected.Four control ones were stopped perfusion.After that,the isolated heart was s cooled down to 10℃,and 4℃cardioplegia(St.Thomas solution or plasma cardioplegia)was perfused at 20 ml/kg.After 30 minutes of hypothermic arrest,it is rewarmed and the K-H solution was perfused again.After 10 minutes equilibration,the time from the beginning of perfusion to heart beating and heart rate was recorded,10 minutes coronary outflow solution was collected.At the end of the experiment,a 3-5 gram sample of myocardium was excised from the suspended heart and fixed in 4% paraformaldehyde(contains 0.1% DEPC)at 4℃for 12-24 h.The tissue was then dehydrated,embedded in paraffin,and sectioned(0.5μm sections) in a standard manner.Another 3-5g sample of myocardium was excised and then frozen rapidly in liquid nitrogen and stored at -70℃.2.Preparation of cold plasma cardioplegia;The blood of mature rabbit is collected through jugular vein intubation and plasma is isolated by method of sedimentation.Mix the plasma with St.Thomas solution by the proportion of 4; 1,and then add potassium to its original concentration.3.Methods of detection; We used high-performance liquid chromatogra-phy and Automatic biochemical apparatus respectively to measure ATP and CK -MB,LD₁/LD₂ content in coronary outflow; TBA(thiobarbituric acid),Pyro-gallpl NBT and DTNB(dithio-bis-nitrobenzoic acid)respectively to measure MDA,SOD and GSH-PX; immunohistochemical study to visualize the pres-ence of TNF,in situ hybridization to visualize the presence and localization of TNF,TUNEL to observe apoptosis,reverse transcription-polymerase chain re-action(RT-PCR)to detect mRNA of Bcl-2.Results1.The time arrested heart by the cold plasma cardioplegia or St.Thomas so-lution was respectively 13.9±2.2 sec or 14.1±2.0 sec,no significant differ-ence P>0.05).2.The reheating time of arrested heart,heart rate,coronary outflow and myocardial water content in group P were respectively 31.8±5.9 sec,128.9±5.2 bpm,5.1±0.2 ml/min and 72%±2%,but in group C they were respec-tively 40.1±4.8 sec,120.3±3.7 bpm,3.3±0.5 ml/min and 77%±14%. There was significant difference between group P and group C(P<0.05).3.CK-MB,LD₁/ LD₂ and ATP content in coronary outflow in group P were respectively 55.64±7.27 IU/L,0.86±0.04 and 0.94±0.17 umol/L, but in group C they were respectively 88.93±3.27 IU/L,1.10±0.10 and 1. 71±0.34 umol/L.There was significant difference between group P and group C P<0.01 or P<0.05).4.MDA and GSH-PX content in myocardium in group P were respectively 0.18±0.03 nmol/mgprot and 27.34±3.03 NU/mgprot,but in group C they were respectively 0.27±0.05 nmol/mgprot and 16.74±1.12 NU/mgprot. There was significant difference between group P and group C(P<0.01).SOD content in myocardium in group P and in group C were respectively 114.15±12.57 NU/mgprot and 104.01±13.87 NU/mgprot,no significant difference P >0.05).5.Immunostaining of TNF protein was not detected in the hearts without I/ R injury.There was more obvious immunostaining in group C than in group P in the hearts undergoing I/R.The expression of TNF mRNA was not detected in the hearts without I/R injury.In situ hybridization of TNF was localized to car-diac myocytes.6.Apoptosis in cardiomyocytes was not observed by TUNEL in the control hearts.In contrast,there were a few apoptosis cardiomyocytes observed by TUNEL after I/R,and TUNEL staining was positive in these cells.7.Bcl-2 mRNA relative content in cardiomyocytes in control group, group P and group C were respectively 0.921±0.015,0.712±0.021 and 0. 504±0.028,with significant difference(P<0.01 or P<0.05).DiscussionIn 1955,Melrose created high potassium cardioplegia.During the following 40 years,myocardial protection,as a key technique,has been developing rapid-ly.St.Thomas solution based on adult animal experiment was used initially in the middle of 70's and is proved to supply perfect protection to mature myocar-dium.But the extrapolation of it to immature myocardium lacks sound basis. Left heart dysfunction caused by inappropriate myocardial protection makes a major reason of postoperative mortality.The structural,functional and metabolic differences between mature and immature myocardium are stressed these days, while generally the same method of protection to them is controversy.1.The feasibility about cold plasma cardioplegic protection to infant rabbit myocardiumNowadays there are a few studies on immature myocardial protection at home and abroad.Some researchers prefer simply hypothermia in immature myo-cardial protection.Others insist that cardioplegia be used as well as hypothermia in order to reduce energy consumption.The perfect cardioplegia can promptly arrest beating heart at its diastolic phase to lead lower oxygen consumption,provide local hypothermia further to demand little energy,keep acid-base balance and stability of cell membrane, supply energy,reduce myocardial edema by its high osmotic pressure.The oxygen consumption of myocardium is lower at hypothermia.It will fall to 39% at 28℃and 50% at 22℃.The cold plasma cardioplegia in this study is composed mainly of plasma,with potassium added.It offers rich metabolic sub-strates,and has enough colloid osmotic pressure thus can relieve cell edema.It has stronger buffer capability and can clear oxygen free radicals.With these the heart energy consumption is low and injury is slight.2.Effects of cold plasma cardioplegia on metabolism of enzyme and energy in infant rabbit myocardiumIn the immature cardiomyocytes there are a few high-energy phosphatase, rich high-energy phosphate,strong ability of breaking down glycogen with or without oxygen.The immature cardiomyocytes is more depended on aerobic or anaerobic metabolism to supply energy.At hypoxemia its anaerobic ability is stronger which can supply more energy because there is a lot of glycogen in the immature cardiomyocytes.In addition,it needs a few energy in contract and possesses strong bearing to acidosis.After reperfusion the myocardial enzyme was leaked out,this was contribu-ted to the change of permeability and integrality of cell membrane.The rate of CK-MB detected out is 100% in the patient with acute myocardial infarct and its specialty is 92%——100%.At the normal condition the LDH spectrum is LD₂>LD₁>LD₃>LD₄>LD₅.LD₁ content in the plasma was increased mainly when the myocardium was injured.This change is earlier than change of LDH. Thus the value CK-MB and LD₁/LD₂ is high sensitive to diagnose myocardial injury.The level of ATP is correlative to state of heart function and effect of my-ocardial protection.The process of myocardial ischemia,in fact,is one of the changes of ener-gy metabolism brought by lack of oxygen and substance to synthesize energy. The local hypothermia can make the immature heart globe hypothermia rapidly, which leads to reduce the myocardial metabolism and arrest beating heart,to protect the myocardium because the small volume,thin wall of ventricle and large ratio of surface and volume of immature heart.Thus the hypothermia is a key role in the.protection of immature myocardium.The system of Na⁺/H⁺ ex-change on cell membrane is important to maintain intracellular pH and cell vol-ume.The cold plasma cardioplegia maybe protect immature myocardium by pro-viding substance to synthesize energy,maintain intracellular stabilization by its strong buffer.3.Effect of cold plasma cardioplegia on oxygen free radicalsMDA is the last product in reaction of lipid peroxidation.It reflects the de-gree of lipid peroxidation in the body,thus reflects indirectly degree of cardio-myocyte injury and quantity of OFR.GSH-PX can clean out harmful metabolic products in cell,and block chain reaction of lipid peroxidation.SOD,a kind of metal proteinase,is an important cleaner of OFR Produced after I/R.It can transform OFR into H₂O₂that is transformed into H₂O by action of GSH-PX.It consumes SOD and GSH-PX to produce a great lot OFR,which causes OFR injury.SOD and GSH-PX can protect structural and functional integrality of cell membrane by cleaning peroxide negative ion.The cold plasma cardioplegia could reduce content of MDA in the immature cardiomyocyte post I/R,and increase content of SOD and GSH-PX,which blocked chain reaction of lipid peroxidation and maintained integrality of cell membrane,to protect immature myocardium.It may be relative to faultiness of Ca²⁺mediated system that there is no significant difference between content of SOD in group P and group C.In addition it may be relative to time of myocardial arrest in this study.4.Effect of cold plasma cardioplegia on apoptosis induced by I/RIt has been proved by recent studies that I/R is an affirmative and complex pathological process that is consist of OFR injury,hydrolyze of ATPase,Ca²⁺ overload and apoptosis.In 1994 Gottlieb first found that I/R could promote cell apoptosis in rabbit heart.Apoptosis is an important part of I/R.Macrophage in myocardium is a main source of TNF,but cardiomyocyte al-so can produce it.TNF produced in cardiomyocyte may be a key factor in im- pacting heart function.TNF as a cytokine promoting inflammation is close rela-tive to cell apoptosis and plays a key role during process of I/R.TNF induced negative inotropic effects after ischemia.The mechanisms by which TNF causes myocardial dysfunction include calcium homeostasis,direct cytotoxicity,oxidant stress,disruption of excitation-contraction coupling,and myocyte apoptosis. TNF induced cardiac myocyte apoptosis by a sphingosine-dependent mecha-nism.TNF activates the sphingomyelin signal transduction pathway,resulting in the production of the intracellular signaling molecule,sphingosine.Since sphin-gosine is a very effective inducer of apoptosis in cardiomyocytes,TNF-induced apoptosis may be mediated by the TNFR1 coupled to the sphingomyelin signal transduction cascade.It also has been shown that TNF induces cardiac myocyte apoptosis via TNF receptor 1.Bcl-2 gene is located 18q21 chromosome.The degree of its expression is relative to Ca²⁺overload,OFR and apoptosis.There is Bcl-2 expression in de-veloping cell,but no or lower express in mature or tending toward apoptosis cell.Many researches show that it can prolong life of cell and prevent apoptosis induced by radial,free radicals and chemical materials,depress affirmatively apoptosis induced by oxidation injury.TNF and Bcl-2 play a key role in myocardial apoptosis induced by I/R.It is important means to prevent or decrease post-ischemic apoptosis that signal transduction cascade of TNF receptor is depressed or blocked and Bcl-2 ex-pression is increased,which are vital methods to prevent or decrease I/R inju-ry.In this study,we found that there was no TNF expression in normal imma-ture myocardium without I/R.After I/R TNF expression is positive,which re-veals I/R can induce cardiomyocyte to produce TNF in no blood circulation. TNF expression was mainly located in cardiomyocyte by immunohistochemical study and in situ hybridization,which reveals cardiomyocyte is a main resource of TNF impacted heart function.Cold plasma cardioplegia could restrain TNF expression in cardiomyocyte by which depressed activation of OFR to P38 mito-gen-activated protein kinase and Nuclear factorκB.In this study,we also found that apoptosis occurs in a part of immature car- diomyocytes and this result suggests that I/R may induce apoptosis in immature myocardium.We also found that I/R may mediate high expression of Bcl-2 which is associated to apoptosis and this result suggests that Bcl-2 system may be involved in the depressing apoptosis.Since the incidence of apoptosis is very low in this study,we did not find the direct effects of cold plasma cardioplegia on apoptosis.This study may have important clinical implications for preservation of the immature myocardium during and after ischemia.The model in this study can provide a convenient and practical way to investigate immature myocardial pro-tection in lab.Conclusion1.Cold plasma cardioplegia could arrest promptly beating heart at diastole.2.Cold plasma cardioplegia was able to increase recovery of coronary out-flow and heart rate and to decrease myocardial edema after I/R.3.Cold plasma cardioplegia lowers CK-MB,LD₁/LD₂ and ATP content in immature myocardial coronary outflow after I/R.4.Cold plasma cardioplegia could depress production of OFR in immature myocardium,and improve antioxidation.5.Cold plasma cardioplegia could restrain TNF expression in cardiomyo-cyte,and enhance Bcl-2 mRNA expression,which restrains apoptosis.