Cardioprotective Effects Of Delayed Preconditioning By Desflurane In Rats

ObjectsEver since the discovery of the phenomenon of cardiac preconditioning, many studies have been done to investigate the mechanism of endogenous protection , which involves nitric oxide (NO) , reactive oxygen species (ROS) , KATP channels, G protein - coupled receptors, opioids, protein kinase C, protein tyrosine kinases, mitogen - activated protein kinases. Delayed preconditioning is a delayed protective adaptation whereby brief episodes of ischemia or pharmacological stimulus enhance the hearts resistance to subsequent ischemia - reperfu-sion injury 24 ~72 h later, so it has greater clinical usefulness.There is increasing evidence to indicated that volatile anesthetics may decrease reversible or irreversible myocardial ischemic injury by delayed preconditioning, apart from NO donor, selective 8 -opioid receptor agonist, adenosine receptor agonist and KATP channel opener. Recently cyclooxygenase - 2 (COX -2) has been found to mediate cardioprotective effects during late preconditioning-This experiment was divided into two parts. Part 1: to investigate whether delayed preconditioning by desflurane has cardioprotective effects, whether COX - 2 mediates its anti - ischemic effect, and influence of selective COX - 2 inhibitor NS -398 on delayed preconditioning by desflurane. Part 2: to investigate the changes of COX - 2 mRNA levels and expression of COX - 2 protein during delayed preconditioning and discuss the mechanism of COX - 2 in delayed preconditioning.MaterialsExperiment animals: 56 Wistar rats, regardless of sex, provided by China Medical University Laboratory Animal Center, weighed between 220 ~300g.Experimental agents and drugs: desflurane (Baxter, USA) , NS -398 and DMSO (Sigma, USA) ; COX -2 monoclonal antibody (Cayman, USA) , pentobarbital sodium ( Shanghai) , 2,3,5 - triphenyltetrazolium chloride ( Shanghai ) , Evans Blue (Shanghai) , normal saline, lactate Ringers solution.Experimental instruments: RM - 6200 polygraph ( Kohden, Japan) , rodent ventilator ( Harvard 638, USA) , Ohmeda - Datex monitor ( USA) , pressure transducer ( Edwards Lifesciences, USA) , Ohmeda 7000 Anesthesia machine (USA), infusor ( B. Braun, Germany), electronic scale (Denver Instrument Company A200 DS, USA) , Jung - CM 1800 Slicer ( Shanghai) , Olympus microscope (Japan) , Heidolph DIAX900 homogenator ( Germany) , PTC - 100 PCR amplification device ( USA) , WFH - 2002 ultraviolet transilluminator (Japan ) , Kodak 1D gel imaging analysis system ( USA) , GIS - 700D digital gel scanning analysis system ( Shanghai).MethodsPart 1: Cardioprotective effects of delayed preconditioning by desflurane in rats32 rats were randomly assigned to one of four groups: control group ( n = 8) , opening of the thorax, occlusion of LAD for 30min and reperfusion for 2h; NS -398 group (n=8), intraperitoneal injection of NS - 398 5mg/kg on day 1, 24h later (day 2) , received LAD occlusion 30min and reperfusion 2h; DES group (n=8), inhaled 1MAC desflurane on day 1, 24h later received same procedure as in NS - 398 group on day 2; NS - 398 + DES group (n=8), intraperitoneal injection of NS -398 5mg/kg on day 1 before inhalation of 1MAC desflurane for 1h, 24h later received same procedure as in NS - 398 group on day 2. After 2h's reperfusion, the hearts were excised and stained using the TTCstaining method. The weight of left ventricle, area at risk (AAR) , infarction region (IS) were measured, and AAR/LV% and IS/AAR% were calculated. Mean arterial pressure ( MAP) , heart rates ( HR) and rate - pressure product (RPP) were recorded at baseline, 30min occlusion, 1h and 2h of reperfusion. Part 2: Influence of delayed preconditioning by desflurane on myocardial expression of COX - 2 .24 rats were randomly assigned to one of three group: control group (n = 8) , after anesthesia by pentobarbital, the hearts were removed; DES group (n= 8) , on day 1 the rats were intubated and mechanically ventilated inhaling lMAC desflurane for lh with respiratory rate 60 bpm and VT5ml approximately. 24h later ( day 2) the hearts were exercised after anesthesia; NS - 398 group (n= 8) , intraperitoneal injection of NS - 398 5mg/kg on day 1 before inhalation of 1MAC desflurane for 1h and received same procedure as in DES group on day 2. After removal of the hearts, left ventricle tissues were separated and stored in liquid nitrogen. RT - PCR and Western blotting were performed to measure myocardial COX - 2 levels and expression of COX - 2 protein.Results1. Hemodynamic changes during ischmia/reperfusion were similar in 4 groups. The changes of MAP, HR, and RPP between groups were not significantly different ( P > 0.05 , respectively).2. No differences were found between groups in body weight, weight of left ventricle and AAR, AAR/LV% ( P >0.05, respectively). IS and IS/AAR decreased significantly in DES group when compared to control group (P < 0. 05 and P <0. 01 respectively) , while no differences were found in NS -398 and NS - 398 + DES group in comparison with control group ( P > 0. 05, respectively ). Weights of IS correlated well with weight of AAR in all four groups with correlation coefficient r =0. 81, 0.78, 0.79 and 0. 89, respectively ( P <0.05 respectively).3. RT-PCR: Myocardial COX -2 mRNA levels in DES group increased markedly compared to control group ( P < 0. 01 ) , while no differences werefound between NS -398 group and control group(P >0.05).4. Western blotting: Compared to control group, myocardial COX -2 expression in DES group increased significantly (P <0.01) , while no differences were found between NS - 398 group and control group( P > 0.05).DiscussionThe mechanism of cardioprotection by volatile anesthetics has been extensively studied. The anti - ischemic effects of volatile anesthetics may be mediated by favorable alterations in myocardial oxygen supply - demand relations, and increased coronary blood flow. But this can be only one of mechanism of cardioprotection by volatile anesthetics, which can not be attributed to the early phase and late phase cardioprotection of late preconditioning by volatile anesthetics.The shift of the heart to a late preconditioned phenotype is the result of a complex cascade of molecular events that begins with an effective preconditioning stimulus (such as reversible ischemia or pharmacological stimuli) and culminates in the synthesis of new proteins that render the heart tolerant to subsequent ischemic injury. The recruitment by the PC stimulus of a series of PKC, PTK and other kinases to the activation of transcription factors that govern the expression of the cardioprotective genes responsible for late preconditioning, which results in the increased tolerance to ischemic injuries and alleviation of degree of myocardial stunning or decrease of myocardial infarction size.Cyclooxygenase - 2 plays an important role in delayed preconditioning and its up - regulation of expression is crucial in mediating cardioprotective effects of delayed preconditioning. The protective effect of COX - 2 against myocardial stunning and infarction is mediated by COX -2 derived PGE2 and PGI2 via enhancement of mitochondrial KATP channel activation.Although COX - 2 was found to mediate ischemic late preconditioning, studies of its involvement in late preconditioning of volatile anesthetics were few. In one study of ischemic late preconditioning found resulted in increase in myocardial COX - 2 mRNA levels followed 24 h later by an increase in COX - 2 protein expression ( over two - fold) and myocardial content of PGE2 and 6 - ke-...