| Objective: To explore the possible mechanism of sevofluranepreconditioning on ischemia reperfusion injury ofthe myocardial protectioneffect of early phase, and provide a theoretical reference forthe clinical application of sevoflurane, by observing the effects of sevofluranepreconditioning on ischemia reperfusion rat myocardial autophagy effect.Method:1Experimental groups and animal modelForty-five clean grade male Sprague–Dawley rats，aged2～3months,weighing200-240g, provided by experimental animal center of HebeiMedical University, were randomly divided into3groups (n=15): sham,operation group (group Sham), ischemia reperfusion group (group I/R),sevoflurane preconditioning group (group Sevo). In group Sham the anteriordescending branch was only exposed but not ligated to observe150min;In group I/R was produced by occlusion of anterior descending branch of leftcoronary artery for30min followed by120min reperfusion in anesthetizedrats to manufacture model of myocardial ischemia reperfusion injury; GroupSevo received15min inhalation of2.5%sevoflurane and15min wash-out30min before myocardial ischemic-reperfusion.All rats were anaesthetized with3%sodium pentobarbital（50mg/kg）viaintraperitoneal injection after8hour fasting. Rats was fixed on thelaboratory bench in supine position. Put hot-water bottle under the trunk andplaced60W incandescent lamp over the trunk to maintain the bodytemperature at37～37.5℃. Limbs connected needle electrodecontinuous testing of lead II electrocardiogram(ECG) by monitor. The ratswere intubated via mouth， ventilated with a rodent ventilator and then connected multiparameter anaesthetic gas monitor to monitor breath. From theneck incision of anterior cervical muscles stripped exposure bronchus, using3-0silk thread ligation in the main bronchus trachea and the endotrachealintubation to avoid aI/R leakage and separating the left carotid arterycannulation to detect arterial pressure. Choracic cavity was exposed via a leftthoracotomy and disconnecting3,4costa at the left edge of0.5cm from themidsternal line after chest sharing and disinfection. Opened the pericardiumto expose the heart,then, the great cardiac vein with the left coronaryartery between left atrial and pulmonary conus was visible. The leftventricular was cannulated through left atrial for development of leftventricular pressure measurement. A6-0Prolene ligature was placed aroundthe proximal left anterior descending coronary artery (LAD) in the proximityof theI/R base. Group Sham, the anterior descending branch was only exposedbut not ligated to observe150min; In group I/R and group Sham wasexperienced occlusion of LAD for30min followed by120min reperfusion,but,group Sevo needed to accept sevoflurane preconditioning before I/R. Theeffectiveness of coronary artery occlusion was verified by epicardial colorchange, the progressive exhibition of marked arrhythmia and the ECG change(QRs wave width increased, sT segment elevated, T wave amplitudeincreased). Successful reperfusion of ischemic myocardium showed: c-olor visual recovery but did not fully recover; contraction compared with theischemia period stronger; QRs wave width and height, sT segment and Twave was significantly decreased, but still higher than the levelbefore ischemia.2Index detection and speciment collection2.1Hemodynamic indexes:Mean arterial pressure (MAP), heart rate (HR), Left ventricular systolicpressure (LVSP) and left ventricular end-diastolic pressure (LVDEP) weremonitored and recorded at10min after atrioventricular canal puncturesucess (T0), ligation of the left coronary artery (T1),30min after occlusioncoronary artery (T2), and10min (T3),30min (T4),1h (T5) and2h(T6) of reperfusion. Then,Calculated and recorded the development of left ventricularpressure (LVDP=LVSP-LVDEP)at every point.2.2Biochemical indexes: Blood sample3ml were taken from the left carotidartery at2h of reperfusion to determine the concentration of cardiac tropine I(cTnI) and creatine kinase-MB (CK-MB).2.3infarction area and autophagy: Ligated Coronary artery again after bloodsampling.5rats of each group were randomly selected to the Evans blue-TTCdouble staining to determine myocardial infarction area. Selected5from theremaining10rats of each group randomly for electron microscope specimens:Tissue samples of the apex cordis, approximately1mm thick, wereimmediately immersed in ice-cold4%glutaraldehyde and preserved at4℃for further processing. Myocardial autophagy occurs were observed usingelectron microscope.2.4The expression of LC3-Ⅱ and Beclin-1: Taken left ventricles ofthe remaining5rats heart of each group to detect myocardial LC3-II andBeclin-1he content by Westeron-Blot method.Result:1The rats’ weight of each group have no statistical difference (P<0.05).2Hemodynamic measurement results2.1Comparision of MAP and HR between the groupsThere was no significant difference in HR at T0～6amonggroups (P>0.1); There was no significant difference in MAP at T0～2amonggroups (P>0.05). Compared with the Sham group, MAP decreased at T3～6inI/R group, and in group Sevo MAP was decreased at T5~6,the difference wasstatistically significant (P<0.05). MAP was significantly increased at T3-6inSevo group compared with group I/R(P<0.05).2.2left ventricular developed pressureThere was no significant difference in LVDP at T0～1amonggroups (P>0.05); LVDP were differences among three groups at T2～6: Compared with Sham group, LVDP was significantly decreased at T2～6in group I/R and Sevo group (P<0.05); Compared with I/R group, LVDPincreased at T2～6in group Sevo,(P<0.05).2.3Comparison of concentration of cTnI and CK-MBCompared with Sham group, the concentration of cTnI and CK-MB inI/R group and Sevo group were significantly increased(P<0.01). Comparedwith the I/R group, the concentration of cTnI and CK-MB in Sevo group weresignificantly decreased (P<0.01).2.4Comparison of myocardial infract areaThe myocardial infarction area in Sevo group (33.5%±8.79%) was lessthan that in I/R group (54.2%±6.34%). The difference was statisticallysignificant (P<0.05).2.5Comparsion of expression of LC3-Ⅱ and Beclin-1Compared with Sham group, expression of LC3-Ⅱ and Beclin-1inI/R group and Sevo group was increased. The difference was statisticallysignificant (P<0.05); There was no significant difference in expression ofLC3-Ⅱ and Beclin-1between group Sevo and group Sham (P>0.05).2.6Changes in myocardial ultrastructureIn group Sham: Myofibrils arranged neatly and sarcomere was clear.Mitochondria and mitochondrial cristae arranged neatly without edema.Autophagic vacuoleslittle is rare to see; The disordered myofibrils,mitochondrial swelling, incomplete mitochondrial membrane, mitochondrialcristae disappeared and a great deal of autophagic vacuoles of differentlevels around the swelling mitochondrial were observed in group I/R.In Sevo group: myocardial fibers arranged neatly compared with I/R groupobviously. Mitochondria swelling significantly reduced. The number ofautophagic vacuoles and lysosomes are reduced a little compared with I/Rgroup.Conclusion:Sevoflurane preconditioning can protect against myocardialischemia/reperfusion (I/R) injury in the acute phase, but, the protective function was independent of the level of autophagy. |
PDF Full Text Request