Sevoflurane Postconditioning Research Articles

Sevoflurane postconditioning protects hearts isolated from rats with diabetes mellitus of different duration against ischemia-reperfusion injury

Objective To investigate the protective effect of sevoflurane postconditioning (Sevo-Postcon)on the hearts isolated from rats with diabetes mellitus (DM) of different duration against ischemia-reperfusion (I/R)injury.Methods Seventy-two pathogen-free male SD rats weighing 200-240 g were randomly divided into 3 groups ( n =24 each):group Ⅰ rats without DM (C) ; group Ⅱ rats with 2 week DM (2w DM) and group Ⅲ rats with 6 week DM (6w DM).DM was produced by intraperitoneal (IP) 1％ streptozocin (STZ) 60 mg/kg and confirmed by fasting blood glucose concentration ＞ 16.7 mmol/L in groups Ⅱ and Ⅲ.Hearts were isolated from rats and perfused with Krebs-Henseleit buffer (KHB) in a Langendorff apparatus.After a 15 min stabilization period,the isolated hearts were subjected to 30 min of global no-flow ischemia followed by 75 min of reperfusion.Twelve hearts in each group were perfused after ischemia with KHB saturated with 3％ Sevo for 15 min followed by perfusion with regular KHB for 60 min.LVEDP,LVDP, ± dp/dt and HR were measured and recorded after 15 min stabilization (T0,baseline) and at 15 and 75 min of reperfusion (T1,2 ).Myocardial specimens were obtained at 15 min of reperfusion (T1) for detection of p-Akt expression (by Western blot analysis).Infarct size was determined at 75 min of reperfusion (T2).Results Sevo-Postcon significantly improved cardiac function,reduced infarct size and up-regulated p-Akt expression in groups Ⅰ (C) and Ⅱ (2w DM),while in group Ⅲ (6w DM) Sevo-Postcon did not cause any change in cardiac function,infarct size and p-Akt expression as compared with the isolated hearts without Sevo-Postcon.Conclusion The cardioprotective effect of Sevo-Postcon can be attenuated with increasing duration of DM by impairing PI3K/Akt signaling pathway. Key words: Anesthetics,inhalation; Diabetes mellitus, experimental; Myocardial reperfusioninjury

Potential Synergy of Antioxidant N -Acetylcysteine and Insulin in Restoring Sevoflurane Postconditioning Cardioprotection in Diabetes

To the Editor: We read with great interest the article recently published by Drenger et al. 1The authors demonstrated in their study that in rats with streptozotocin-induced type 1 diabetes, the cardioprotective effects of either ischemic postconditioning or the volatile anesthetic sevoflurane postconditioning were lost and that short duration (48 h) of insulin treatment to “normalize” glucose concentration failed to restore the sensitivity of the hearts from early-stage (4–5 weeks after streptozotocin injection) diabetic rats to postconditioning. Of interest, the author showed that insulin administration before ischemia–reperfusion event in the model of diabetic rats exacerbated postischemic myocardial infarction than without insulin therapy. Accordingly, they suggested that caution should be taken not to add insulin before the planned ischemic period.However, we cannot completely agree with the authors' suggestion. Hyperglycemia-induced increase in mitochondrial superoxide anion production has been shown to represent a key mechanism of hyperglycemic damage.2Results of prospective, randomized clinical trails have shown that aggressive control of blood glucose achieved by intensive insulin therapy (target glucose concentration of 80–110 mg/dl) significantly decreased mortality in critically ill patients3whereas poor or marginal intraoperative blood glucose control was associated with a worse hospital outcome after cardiac surgery in diabetic patients.4In the study by Drenger et al. ,1the blood glucose concentrations in the diabetic insulin-treated rats receiving postconditioning (mean 126 ± 10 mg/dl and 131 ± 9 mg/dl, respectively, in the ischemic and sevoflurane postconditioning groups) were at most marginally controlled. Given the big SDs and small sample size in these groups (n = 6 per group), the glucose concentration in a considerable number of rats in these groups could have been hyperglycemic rather than normoglycemic (defined by the authors as lower than 135 mg/dl). Therefore, it seems reasonable for us to postulate that it was the insufficient acute insulin therapy but not the administration of insulin before ischemia that resulted in the exacerbation of postischemic infarct size seen in the study of Drenger et al. 1The exact mechanism for this phenomenon is unclear. One possible explanation, in addition to those postulated by the authors, could be that insufficient acute insulin therapy may have abrogated chronic hyperglycemia-induced compensatory increases of myocardial endogenous antioxidant enzymes such as superoxide dismutase enzyme production and activity5and rendered the diabetic hearts more vulnerable to oxidative damage after ischemia–reperfusion.We want to congratulate the authors for their in-depth mechanistic explorations, which showed that phosphorylation of the signal transducer and activator of transcription 3 (STAT3), a key mediator in postconditioning-mediated cardioprotection,6was reduced in various parts of the diabetic myocardium (which may contribute to the diabetes-related loss of ischemic and sevoflurane postconditioning). This is an important finding that suggests that enhancing myocardial p-STAT3 may restore postconditioning cardioprotection in diabetes. Indeed, in a similar model of streptozotocin-induced diabetic rats, we found that myocardial p-STAT3 was significantly reduced in rats 4–5 weeks after streptozotocin injection and that treatment with the antioxidant N -acetylcysteine enhanced myocardial p-STAT3 and attenuated postischemic myocardial infraction.7Furthermore, we showed in the same model of diabetic rats that N -acetylcysteine treatment restored the responsiveness of diabetic hearts to anesthetic preconditioning with remifentanil.8The findings by Drenger et al. 1and us7,8together with the findings of Kehl et al. , who showed that N -acetylcysteine can restore isoflurane-induced preconditioning against myocardial infarction during hyperglycemia,9lead us to postulate that N -acetylcysteine may restore sevoflurane postconditioning in diabetes, possibly by enhancing myocardial p-STAT3. Given that short duration of N -acetylcysteine treatment did not reduce plasma glucose concentration in diabetic rats,7we further postulate that N -acetylcysteine and insulin may potentially confer synergy in restoring sevoflurane postconditioning cardioprotection in diabetes.In summary, we agree with the authors that a longer period of insulin therapy might have a different effect on restoring postconditioning in diabetes. However, we propose that prompt insulin administration, in particular sufficient insulin therapy when added before the planned ischemic period, should also confer beneficial effects, especially when it is used in conjunction with other therapy such as antioxidants, although this hypothesis need to be further tested.

The Effect of Insulin May Not Be So Simple

To the Editor: Drenger et al. have demonstrated that sevoflurane postconditioning is cancelled in the rat heart with type 1 diabetes mellitus and that this adverse effect by glucose intolerance cannot be restored by the adjustment of blood glucose using insulin.1This study appears to have many questions regarding the mechanisms of insulin's action, whereas we would congratulate their impressive results. In the study by Drenger et al. , the treatment with insulin significantly increased the infarct size in rats with diabetes mellitus, and a phosphatidylinositol-3-kinase inhibitor wartmannin demonstrated the effect to a similar extent.1As Drenger et al. have mentioned in the Discussion section, this phenomenon is difficult to explain1because insulin is a well-known phosphatidylinositol-3-kinase activator in the cardiac myocytes.2As a previous elegant study showed that diabetes abolishes the morphine-induced postconditioning effect in the rat heart, evaluation of the related pathways, including glycogen synthase kinase 3β, janus-activated kinase, signal transducer and activator of transcription 1, phosphatidylinositol-3-kinase/Akt, and extracellular signal-related kinase, in addition to signal transducer and activator of transcription 3, would help in understanding the study by Drenger et al. 1,3Diabetes mellitus may down-regulate a redox sensitive transcription factor, NF-E2-related factor 2 activity via extracellular signal-related kinase, resulting in impairment of the sevoflurane postconditioning effect because this pathway has been proved to be induced by the oxidative stress in the diabetic heart.2We also have to keep in mind that 5-hydroxydecanonate is not a selective inhibitor of mitochondrial adenosine triphosphate sensitive K+channels anymore because it is a substrate for the enzyme acyl-CoA synthethase in the electron transport chain of mitochondria,4and it is capable of playing a role as an inhibitor of sarcolemmal adenosine triphosphate sensitive K+channels.5Therefore, further studies are needed to clarify the mechanistic role of insulin in relation to diabetes mellitus on the sevoflurane postconditioning effect toward the ischemic heart.

In Reply

Hadassah-Hebrew University Medical Center, Jerusalem, Israel. drenger@cc.huji.ac.ilI have read with great interest the comments of Drs. Liu and Xia. Their study on a similar model of streptozotocine-induced diabetic rats1confirmed my findings that myocardial signal transducer and activator of transcription 3 (STAT3) concentrations are significantly decreased in the diabetic heart, whereas normal STAT3 concentrations are essential for initiation of the protective process of sevoflurane postconditioning (postC) in diabetes. They also described their findings that STAT3 concentrations might recover in diabetic hearts in the presence of N -acetylcysteine. They suggest that N -acetylcysteine and allupurinol promote tolerance of the myocardium to ischemic injury by restoring adinopectin concentration, and thus, they propose that N -acetylcysteine might be effective in restoring sevoflurane postC in the diabetic heart.The animal model used in the study2was type I diabetes, i.e. , uncorrected hyperglycemia for 1 month. Insulin was given in the last 48 h to bring the animals into a state of normoglycemia at the time of the experiment. I did not give insulin therapy to alleviate the symptoms of diabetes, but to perform the experiments in a normoglycemic state, with a goal to reduce blood sugar to less than 135 mg/dl, but I actually achieved better control as reflected in the study. The diabetic irresponsiveness of the myocardium is more complex than that expected by the hyperglycemic state alone. It might be attributed to multiple cellular changes in mitochondrial membrane potentials, which lead to certain degrees of mitochondrial uncoupling.3This in turn leads to a glycation reaction, causing cellular injury and accumulation of glycation end products,4depression of more signal transduction components such as phosphatidylinositol 3-kinase, STAT3,2and nitric oxide synthase, as well as glycogen synthase kinase-3β activation,5all leading to lipid accumulation, inflammation, and remodeling.6Therefore, I do not agree with the comment that strict preservation of normoglycemia during the experiment might eliminate all changes accumulated during the month of diabetic state. The changes in the heart are too substantial to be prevented by a single maneuver of replenishing antioxidant state.From a practical view, giving higher doses of insulin to an animal would be irresponsible, because hypoglycemia in animals who are not observed closely most of the time would be detrimental. I am not aware of any animal study evaluating diabetes for long-term outcome that used conventional glucose control levels. Extrapolating from human data that use conventional glucose control, as suggested in the current letter to the editor, on patient postoperative long-term outcome, is controversial at best. Strict control of diabetes at normal levels was found hazardous due to severe side effects.7,8Attributing changes in sensitive cellular pathways, such as STAT3 and phosphatidylinositol 3-kinase, only to the natural lack of antioxidants seems simplistic. As discussed in the previous paragraph, diabetes effects on the heart are profound and the mechanism of injury is at multiple levels.The authors claim that a longer period of insulin therapy might have a beneficial effect on restoring postC in diabetes. Unfortunately, I suggested differently, that changes in the heart caused by diabetes were not restored by a short period of normoglycemia. Insulin administration before complete ischemia may carry the risk of enhanced cellular metabolism in the no-flow state, a situation that may bring about accumulation of high tissue levels of lactate, which inhibits glycolysis and prevents any glycolytic stimulation by insulin.9,10Therefore, until proved experimentally, I cannot agree with the authors' final statement that insulin administration before ischemia may produce beneficial effects.

Myocardial protective effect of sevoflurane postconditioning and its mechanism

Background Many evidences have showed that mechanical ischemic and pharmacological postconditioning are protective against myocardial ischemia/reperfusion injury.Sevoflurane,a novel and ideal volatile anesthetic,is most commonly used to maintain the state of general anesthesia.Experiments have proved that sevoflurane postconditioning can protect against myocardial ischemia/reperfusion injury(I/RI).Objective The review elaborates myocardial protection of sevoflurane postconditioning and its mechanism by summarizing studies and researches in recent years.Content Sevoflurane postconditioning can reduce reperfusion myocardial infarct size,mitochondrial damage and ventricular arrhythmia,as well as improve cardiac hemodynamic after reperfusion.The myocardial protection mechanism involves blocking mitochondrial permeability transition pore,activating mitochondrial KATP-Channels(mKATp),extracellular signal-regulated kinase 1/2(ERK 1/2)and phatidylinosito1-3-kinase-serine/threonine kinase(PI3K-Akt)signaling pathway,etc.Trend Researches in future should be taken to further explore of the mechanism of sevoflurane postconditioning.And strengthening the clinical applications of sevoflurane postconditioning will also be helpful to provide a reliable basis for the practice work. Key words: Sevoflurane; Ischemia postconditioning; Ischemia/reperfusion injury; Myocardial protection

The effect of sevoflurane postconditioning on cardioprotection against ischemia-reperfusion injury in rabbits

Sevoflurane postconditioning is a potential clinical measure to protect myocardial. This experiment was designed to investigate the efficacy of sevoflurane postconditioning against ischemia-reperfusion injury. A total of 132 Japanese White Rabbits were enrolled into this study. They were underwent 15-, 30-, or 60-min left anterior descending coronary (LAD) artery occlusion, respectively. At the end of LAD artery occlusion, they randomly received a 5-min inhalation of air (control group), 1% sevoflurane (1% sev group), 2% sevoflurane (2% sev group), 4% sevoflurane (4% sev group) or an IV bolus injection of 5mg/kg of NIM811 [a specific inhibitor of mitochondrial permeability transition pores (mPTP)]. Infarct size was determined after 2h of reperfusion (triphenyltetrazolium chloride straining, percentage of risk area). The infarct sizes were significantly (P<0.05) reduced after 15min ischemia (5.5±3.3%, 5.8±3.6% vs. 20.3±6.9% for 2% sev, 4% sev vs. control, respectively) and 30min ischemia (23.5±5.0%, 20.7±5.9% vs. 50.9±10.2%, for 2% sev, 4% sev vs. control, respectively; P<0.05). However, it had no effect on infarct size after 60min ischemia (64.1±5.9%, 62.3±7.6% vs. 72.7±9.2% for 2% sev, 4% sev vs. control, respectively, P>0.05).The efficacy of sevoflurane postconditioning gradually weakened with increasing ischemia duration and disappears after 60min ischemia in rabbits in vivo.

ORAL PRESENTATION

ORAL PRESENTATION

Sevoflurane postconditioning attenuates myocardial apoptosis in isolated rat hearts via a modulation of Bcl-2 family proteins

To explore the effects of sevoflurane postconditioning on ischemic/reperfused myocardial apoptosis. Isolated perfused rat hearts were randomly assigned into 3 groups: sham-operation (sham), ischemia/reperfusion (I/R) and sevoflurane postconditioning (SPC). Except for the sham group, the hearts were subjected to 40 min global myocardial ischemia and 120 min reperfusion. Left ventricular systolic pressure (LVSP), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximum increase rate of LVDP (+dp/dt), maximum decrease rate of LVDP (-dp/dt), heart rate (HR) and coronary flow (CF) were measured at baseline, R (reperfusion) 30 min, R60 min, R90 min and R120 min. Creatine kinase (CK) and lactate dehydrogenase (LDH) were measured at 5 min and 10 min post-reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining at the end of reperfusion. The expressions of Bcl-2 and Bax were determined by Western blot. The values of LVSP, LVDP, ± dp/dt and CF were higher while that of LVEDP was lower in the SPC group than the I/R group at all time points of reperfusion (P < 0.05). The releases of CK and LDH and infarct size were significantly reduced in the SPC group versus the I/R group (22.2% ± 2.8% vs I/R: 44.9% ± 6.6%, P < 0.05). The expression of Bcl-2 increased significantly while that of Bax decreased in the SPC group verus the I/R group. Sevoflurane postconditioning may improve myocardial functions, reduce infarct size and attenuate myocardial apoptosis. And the modulated expression of apoptotic proteins plays an important role in sevoflurane-induced myocardial protection.

Diabetes Blockade of Sevoflurane Postconditioning Is Not Restored by Insulin in the Rat Heart

The possibility of restoring sevoflurane postconditioning (sevo-postC) cardioprotection in diabetic animals is uncertain. We hypothesized that attenuation of myocardial injury by sevo-postC might be hindered by inhibition of signal transducer and activator of transcription (STAT) 3-regulated activity of phosphatidylinositol 3-kinase (PI3K) in diabetic animals. To determine whether postC cardioprotection can be restored by normoglycemia, we treated rats with insulin. Diabetic or nondiabetic rats were randomly subjected to 30-min ischemia/reperfusion, with ischemic postC or sevo-postC, with and without mitochondrial adenosine triphosphate-dependent potassium channel blocker 5-hydroxy decanoate sodium and PI3K antagonist wortmannin. The infarct area, phosphorylated STAT3, and apoptosis were examined. Studies were repeated after insulin treatment. Ischemic postC and sevo-postC significantly reduced infarct size by 50% in the nondiabetic rats (P < 0.002), a phenomenon completely reversed by 5-hydroxy decanoate sodium and wortmannin. Diabetes mellitus blocked the protective effect of postC, and insulin treatment to achieve normoglycemia did not restore cardioprotection. Phosphorylated STAT3 nuclear retention was significantly increased after ischemia-reperfusion and was further enhanced in response to ischemic postC (P < 0.05) but was significantly reduced in diabetic rats (by 43%; P < 0.01). The effective reduction in infarct size and apoptosis in the nondiabetic rat heart by postC was completely abrogated in diabetic rats. This inhibition is not relieved by insulin-induced normoglycemia. The PI3K pathway and mitochondrial adenosine triphosphate-dependent potassium channel activation are involved in the mechanism of postC. In diabetic rats, STAT3 activation was strongly reduced, as was postC cardioprotection, suggesting that the inability of insulin to restore postC may be attributed to diabetes-induced STAT3-mediated inhibition of PI3K signaling.

Gender-Related Difference of Sevoflurane Postconditioning in Isolated Rat Hearts: Focus on Phosphatidylinositol-3-Kinase/Akt Signaling

Previous studies have reported that female gender confers cardioprotection against ischemia/reperfusion (I/R) injury, partly because estrogen activates phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. We have previously proven that cardioprotection of sevoflurane postconditioning is mediated by PI3K/Akt pathway in male rats. The purpose of the present study was to determine whether the cardioprotection of sevoflurane postconditioning is influenced by gender, and the role of PI3K/Akt pathway in such gender difference. Isolated hearts from 2-mo-old male and female SD rats were subjected to ischemia for 40 min and reperfusion for 2 h in the Langendorff apparatus, and were randomly assigned to the following groups: no ischemia/reperfusion (CON), ischemia/reperfusion (I/R), I/R+sevoflurane postconditioning (I/R+SPC), I/R+100 nM wortmannin (I/R+WOR), and I/R+SPC+WOR. Postconditioning was performed with administration of 3.0% sevoflurane at the first 10 min of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and myocardial lactate dehydrogenase (LDH) release were measured. Infarct size was detected by riphenyltetrazolium chloride staining. The protein expression of total Akt (t-Akt) and phosphorylated Akt (Ser(473)) (p-Akt) were determined by Western blot. The I/R group showed lower LVDP and higher LVEDP than CON group in the same gender during reperfusion period. The LDH release and infarct size were smaller in the female I/R group (P < 0.05 versus male I/R group). Sevoflurane postconditioning markedly improved left ventricular function and decreased LDH, infarct size in the male I/R+SPC group (P < 0.05 versus male I/R group) but not in the female I/R+SPC group. Wortmannin abolished the cardioprotection of sevoflurane postconditioning in the male I/R+SPC+Wort group (P < 0.05 versus male I/R+SPC group), and markedly increased the infarct size and LVEDP and decreased LVDP in female rats. The t-Akt protein expression was no significant difference in all groups. The ratio of p-Akt/t-Akt expression in the male CON group was a little lower than that in the female CON group, but there was no statistical significance. In male rats, the ratio of p-Akt/t-Akt was no difference between CON and I/R group, but it was higher in I/R+SPC group than that in I/R group (P < 0.05). In female rats, the level of p-Akt was markedly increased by I/R, which was markedly higher than that in male I/R group (P < 0.05). However, p-Akt was not different between I/R and I/R+SPC groups. Wortmannin decreased the p-Akt expression in both male and female rats. It is concluded that female rat hearts showed greater resistance to I/R injury, and sevoflurane postconditioning developed cardioprotection in male rats but not in female rats. The PI3K/Akt pathway may be involved in the cardioprotection by both sevoflurane postconditioning and gender.

The effects of fentanyl combined with sevoflurane postconditioning on myocardial functions of isolated ischemia-reperfused rat heart

Objective To investigate the effects of fentanyl combined with sevoflurane postconditioning on myocardial functions of isolated ischemia-reperfused rat hearts.Methods Depending on the different treatments given at 10 min after reperfusion started,isolated perfused rat hearts were randomly assigned to the following four groups(n=10):ischemia/reperfusion (Con),sevoflurane postconditioning (SPC),fentanyl postconditioning (Fent),and fentanyl plus sevoflurane postconditioning (Fent+SPC). All of the hearts were subjected to 40 min global myocardial ischemia and 120 min reperfusion. Left ventricular systolic pressure(LVSP),Left ventricular developed pressure (LVDP),maximum increasing rate of LVDP(+dp/dt),maximum decreasing rate of LVDP (-dp/dt),heart rate (HR) and coronary flow (CF) were recorded. Lactate dehydrogenase(LDH) was measured. Infarct size was determined by triphenyltetrazolium chloride staining.Results LVSP,LVDP,±dp/dt,and CF were higher in SPC group than in Con group at all points of reperfusion (P＜0.05). Compared with SPC group,the Fent and Fent+SPC group (P＜0.05) had significantly improved LVSP and LVDP at the beginning 15 min of reperfusion,which were gradually reduced and lower than SPC group at all time points after 60 min of reperfusion. There were no significant diffirences in HR among four groups. The values of ±dp/dt and CFin Fent and Fent+SPC group were dramatically improved in the early stage of reperfusion(P＜0.05),while similar to the Con group at the end of reperfusion. The SPC,Fent and Fent+SPC group had a significantly reduced LDH release and decreased infarct size compared with the Con group [(22.7±2.3)%, (20.6±2.8)%, (21.3±3.3)% vs.(45.8±3.2)%,respectively; P＜0.05).Conclusion Sevoflurane postconditioning improves myocardial Key words: Sevoflurane; Fentanyl; Postconditioning; Ischemia/Reperfusion; Myocardial function

Sevoflurane Postconditioning Ameliorates Oxygen–Glucose Deprivation—Reperfusion Injury in the Rat Hippocampus

Sevoflurane is well known to exert a neuroprotective effect through anesthetic preconditioning. However, its effects on postconditioning, a neuroprotective phenomenon following an insult, have not been well studied. In this study, we examined the ability of sevoflurane to induce postconditioning in rat hippocampal slices, in vitro. 2%, 4%, and 6% sevoflurane reduced neurophysiologic and morphologic neuronal injury following oxygen-glucose deprivation (OGD) and reperfusion. The quantity of damaged neurons was significantly reduced on immunofluorescence staining; excitatory amino acids (Asp, Glu) increased and inhibitory amino acids (GABA) decreased significantly. The effect was concentration-dependent. Postconditioning with sevoflurane reduces neuronal damage after OGD-reperfusion injury in the CA1 area of rat hippocampus, in vitro.

Effects of sufentanil postconditioning and sevoflurane postconditioning on myocardial ischemia/reperfusion injury in isolated rat hearts

Objective To investigate the effects of sufentanil postconditioning and sevoflurane postconditioning on myocardial ischemia/reperfusion (I/R) injury in isolated rat hearts. Methods Healthy male SD rats weighing 230-250 g were anesthetized with intraperitoneal 5% chloral hydrate 8 ml/kg. Their hearts were excised and perfused in a Langendorff apparatus with modified K-H solution saturated with 95% O2-5% CO2 at 37℃. Forty isolated rat hearts with I/R injury were randomly assigned into 4 groups ( n = 10 each): Ⅰ I/R group, Ⅱ sevoflurane postconditioning group, Ⅲ sulfentanil postconditioning group and Ⅳ postconditioning with sevoflurane and sulfentanil group. After 30 min of stablization, the hearts were subjected to 40 min of global ischemia followed by 120 min of reperfusion. Fifteen minutes of drug postconditioning were performed in group Ⅱ , Ⅲ and Ⅳ:3.0% sevoflurane was introduced into K-H solution in group Ⅱ , 100 nmol/L sulfentanil was added to K-H solution in group Ⅲ , sevoflurane postconditioning and sulfentanil postconditioning were performed simultaneously in group Ⅳ . Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure ( LVEDP), left ventricular developed pressure (LVDP), maximum increase in rate of LVDP ( + dp/dtmax), maximum decrease in rate of LVDP ( - dp/dtmax), HR and coronary flow (CF) were measured at the end of 30 min stablization (baseline), and at 15, 30, 60, 90 and 120 min of reperfusion. Coronary effluent was collected at 5 min of reperfusion for determination of activities of creatine kinase (CK) and lactate dehydrogenase (LDH). Myocardial tissues were obtained at the end of reperfusion for determination of infarct size (by triphenyltetrazolium chloride staining) and expression of Bcl-2 and Bax (by Western blot). The ratio of Bcl-2/Bax was calculated. Results LVSP, LVDP, ± dp/dtmax,CF, Bcl-2 expression and ratio of Bcl-2/Bax were significantly higher, LVEDP, activities of CK and LDH and Bax expression were significantly lower, and the infarct size was smaller in group Ⅱ , Ⅲ and Ⅳ than in group Ⅰ (P ＜ 0.05 or 0.01 ). There were no differences in the indices mentioned above between Ⅱ , Ⅲ and Ⅳ groups.Conclusion Sufentanil postconditioning can attenuate myocardial I/R injury, and there is no enhancement of myocardial protection when combined with sevoflurane postconditioning. The mechanism of myocardial protection is related to the inhibition of cell apoptosis via up-regulation of Bcl-2 expression and down-regulation Bax expression. Key words: Sufentanil; Myocardial reperfusion injury; Sevoflurane; Ischemic postconditioning

Sevoflurane postconditioning reduces myocardial reperfusion injury in rat isolated hearts via activation of PI3K/Akt signaling and modulation of Bcl-2 family proteins

Sevoflurane postconditioning reduces myocardial infarct size. The objective of this study was to examine the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic postconditioning and to determine whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in sevoflurane postconditioning. Isolated and perfused rat hearts were prepared first, and then randomly assigned to the following groups: Sham-operation (Sham), ischemia/reperfusion (Con), sevoflurane postconditioning (SPC), Sham plus 100 nmol/L wortmannin (Sham+Wort), Con+Wort, SPC+Wort, and Con+dimethylsulphoxide (DMSO). Sevoflurane postconditioning was induced by administration of sevoflurane (2.5%, v/v) for 10 min from the onset of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximum increase in rate of LVDP (+dP/dt), maximum decrease in rate of LVDP (-dP/dt), heart rate (HR), and coronary flow (CF) were measured at baseline, R30 min (30 min of reperfusion), R60 min, R90 min, and R120 min. Creatine kinase (CK) and lactate dehydrogenase (LDH) were measured after 5 min and 10 min reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining at the end of reperfusion. Total Akt and phosphorylated Akt (phospho-Akt), Bax, Bcl-2, Bad, and phospho-Bad were determined by Western blot analysis. Analysis of variance (ANOVA) and Student-Newman-Keuls' test were used to investigate the significance of differences between groups. The LVDP, + or - dP/dt, and CF were higher and LVEDP was lower in the SPC group than in the Con group at all points of reperfusion (P<0.05). The SPC group had significantly reduced CK and LDH release and decreased infarct size compared with the Con group [(22.9 + or - 8)% vs. (42.4 + or - 9.4)%, respectively; P<0.05]. The SPC group also had increased the expression of phospho-Akt, Bcl-2, and phospho-Bad, and decreased the expression of Bax. Wortmannin abolished the cardioprotection of sevoflurane postconditioning. Sevoflurane postconditioning may protect the isolated rat heart. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play an important role in sevoflurane-induced myocardial protection.

Role of PI3K/Akt signal pathway in sevoflurane postconditioning against focal cerebral ischemia-reperfusion injury in rats

Objective To investigate the role of PI3KIAkt signal pathway in sevoflurane postconditioning against focal cerebral ischemia-reperfusion （I/R） injury. Methods Sixty-four adult male SD rats weighing 300- 350 g were randomly assigned into 4 groups （ n = 16 each） ： group I focal cerebral I/R （group I/R） ; group II sevoflurane postconditioning （group SP） ; group III sevoflurane postconditioning ＋ wortmannin （group SP ＋ W） and group IV I/R ＋ wortmannin （group I/R ＋ W） . The animals were anesthetized with intraperitoneal 10% chloral hydrate 300-350 mg/kg, traeheostomized and mechanically ventilated. ECG was continuously monitored. Right femoral artery was cannulated for BP monitoring. Focal cerebral I/R was induced by electro-coagulation of left middle cerebral artery and 60 min occlusion of bilateral common carotid arteries followed by reperfusion. The animals inhaled 2.5% sevoflurane in 02 for 60 min as soon as reperfusion was started in group II and III . Wortmannin 0.6 mg/kg was administered iv at 30 rain before reperfusion in group III and/ IN. Neurological function was assessed and scored at 24, 48 and 72 h （T1-3 ） of reperfusion. The animals were decapitated after the last neurological function assessment. Their brains were immediately removed for determination of infarct size. The expression of phosphorylated Akt and Bad （p-Akt, p-Bad） were determined by Western-blot analysis in isehemic penumbra. Results Sevoflurane postconditioning significantly improved the neurological function, decreased the infarct size and increased the expression of p-Akt and p-Bad in group II as compared with group I . There was no significant difference in all variables between group I and group IV . Wortmannin attenuated the protective effect of sevoflurane postcoditioning against focal cerebral I/R injury. Conclusion Up-regulation of p-Bad expression induced by activation of PI3K/Akt signal pathway is involved in the protective effect of sevoflurane postconditioning against focal cerebral I/R injury. Key words: Anesthetics, inhalation ; Brain ischemia ; Reperfusion injury ; Postconditioning

Attenuation of ischemia–reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts

Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.

Sevoflurane postconditioning prevents activation of caspase 3 and 9 through antiapoptotic signaling after myocardial ischemia–reperfusion

Volatile anesthetic postconditioning reduces apoptosis through antiapoptotic signaling. Whether sevoflurane postconditioning prevents activation of caspase 9 and 3, which are implicated in the initiation and execution step of apoptosis, is not known. Isolated perfused guinea pig hearts underwent 30 min global ischemia and 120 min reperfusion [control (CTL)]. Anesthetic postconditioning was elicited with sevoflurane (2%) for 2 min at reperfusion onset (POST). LY294002, phosphatidylinositol-3-kinase (PI3K)/Akt (protein kinase B) inhibitor; and PD98059, extracellular signal-regulated kinase 1/2 (ERK) inhibitor, were administered for 10 min before ischemia and throughout the reperfusion period in POST (POST + LY, POST + PD). Left-ventricular-developed (LVDP) and LV end-diastolic (LVEDP) pressures and infarct size were measured. Western blot analysis determined phosphorylated Akt and ERK expression. Myocardial caspase 3 and 9 were determined immunohistochemically. After ischemia-reperfusion, POST had higher LVDP (57 +/- 9 vs. 38 +/- 7 mmHg, p < 0.05) and lower LVEDP (21 +/- 8 vs. 46 +/- 15 mmHg, p < 0.05) versus CTL. Infarct size was significantly reduced in POST versus CTL (15 +/- 3 vs. 41 +/- 11%, p < 0.001). Phosphorylation of Akt and ERK after reperfusion was significantly increased in POST versus CTL. Immunoreactivity for caspase 3 and 9 was greater in the nucleus of myocytes and endothelial cells in CTL. POST attenuated this increased immunoreactivity. LY294002 and PD98059 abolished the effect of POST on caspase 3 and 9 immunoreactivity. Sevoflurane postconditioning prevents activation of caspase 3 and 9, mediators of apoptosis in ischemia-reperfusion injury. This caspase activation is mediated by phosphorylation of Akt and ERK.

セボフルランによるポストコンディショニングは虚血再灌流後の抗アポトーシスシグナル伝達経路を介してカスパーゼ3, 9 の活性を妨げる

セボフルランによるポストコンディショニングは虚血再灌流後の抗アポトーシスシグナル伝達経路を介してカスパーゼ3, 9 の活性を妨げる

Sevoflurane Postconditioning Attenuates Spinal Cord Reperfusion Injury Through Free Radicals-Mediated Up-Regulation of Antioxidant Enzymes in Rabbits

Paraplegia is one of the most common complications following aortic aneurismal surgery. The present study examined the hypothesis that neuroprotection of sevoflurane postconditioning (PostC) against spinal cord reperfusion injury is associated with free radicals-mediated up-regulation of antioxidant enzymes in rabbits. New Zealand White rabbits were subjected to sevoflurane PostC, oxygen (O(2)), or sham treatment at the onset of spinal cord reperfusion for 10 min. Spinal cord ischemia was induced by clamping the aorta both below the left renal artery and above the aortic bifurcation. Forty-eight hours after reperfusion, the motor function of the lower limbs was evaluated and the lumbar spinal cord segment (L5-7) was harvested for histopathologic and biochemical analyses in the presence or absence of N-2-mercaptopropionylglycine, a potent oxygen free radical scavenger. Activities of superoxide dismutase and catalase in spinal cord tissue were increased in the sevoflurane PostC group 1 h after reperfusion and retained a higher level 6 or 24 h after reperfusion. Sevoflurane PostC produced a significant reduction of the malondialdehyde content at 6, 24, and 48 h after reperfusion. A 20 min continuous administration of N-2-mercaptopropionylglycine starting 10 min before the beginning of PostC attenuated the neuroprotective effect against spinal cord ischemia, and reversed the increase of the two enzyme activities in spinal cord tissue induced by sevoflurane PostC. An initial oxidative stress, as a trigger to up-regulate the antioxidant enzyme activities, plays an important role in the protection of sevoflurane PostC against spinal cord reperfusion injury.

Aprotinin Abolishes Sevoflurane Postconditioning by Inhibiting Nitric Oxide Production and Phosphorylation of Protein Kinase C-δ and Glycogen Synthase Kinase 3β

It remains controversial whether aprotinin use during cardiac surgery is cardioprotective or detrimental. In contrast, volatile anesthetics may offer cardioprotection perioperatively. Increased nitric oxide, protein kinase C activation, and glycogen synthase kinase 3beta inhibition play a role in sevoflurane-induced cardioprotection. The authors investigated whether aprotinin affects sevoflurane postconditioning. Isolated guinea pig hearts underwent 30 min of global ischemia and 120 min of reperfusion (control [CTL]). Postconditioning was elicited with sevoflurane (2%) for 2 min at reperfusion onset (POST). Aprotinin (250 kallikrein inhibitor units/ml) was administered for 5 min at reperfusion onset (POST + APRO and CTL + APRO). In additional experiments, both sevoflurane and aprotinin were given before ischemia and throughout the reperfusion period (SEVO + APRO (throughout)) to mimic clinical conditions. Left ventricular developed and end-diastolic pressures and infarct size were measured. Western blot analysis determined phosphorylated protein kinase C-delta, protein kinase C-delta, Akt, and glycogen synthase kinase 3beta expression. Nitric oxide production during reperfusion was measured by nitric oxide sensor. After ischemia-reperfusion, POST had significantly higher left ventricular developed (56 +/- 11 vs. 26 +/- 8 mmHg [mean +/- SD]) and lower end-diastolic pressures (20 +/- 9 vs. 47 +/- 15 mmHg) and reduced infarct size (15 +/- 3% vs. 41 +/- 10%) versus CTL. Aprotinin abolished these improvements. Expressions of phospho-Akt (activated), phospho-protein kinase C-delta (activated), and phospho-glycogen synthase kinase 3beta (inhibited) were significantly increased in POST. Aprotinin attenuated these increased expressions. Nitric oxide production after reperfusion was higher in POST than in CTL, but not in POST + APRO. Aprotinin abolishes sevoflurane postconditioning, associated with inhibited phosphorylation of Akt, protein kinase C-delta, and glycogen synthase kinase 3beta and reduced nitric oxide production.