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Objective To evaluate the relationship between the mechanism underlying inhibition of inflammatory responses induced by α7 nicotinic acetylcholine receptor(α7nAChR)agonist postcondition?ing alone or in combination with remote limb ischemic postconditioning during myocardial ischemia?reperfu?sion(I∕R)and glycogen synthase kinase?3β(GSK?3β)in rats. Methods Eighty adult male Sprague?Dawley rats, aged 8 weeks, weighing 290-320 g, were divided into 4 groups(n=20 each)using a ran?dom number table: I∕R group, α7nAChR agonist postconditioning group(group P), remote limb ische?mic postconditioning group(group L)and α7nAChR agonist postconditioning plus remote limb ischemic postconditioning group(group P+L). Myocardial I∕R was induced by 30 min occlusion of the left anterior descending branch of coronary artery followed by 120 min reperfusion. Specific α7nAChR agonist PNU282987 2 mg∕kg was intravenously injected immediately before reperfusion in group P. In group L, limb ischemia was induced by tourniquet occlusion of bilateral hind paws for 10 min starting from 20 min of myocardial ischemia, and the tourniquet was released at the beginning of reperfusion. Combination of inter?vention measures previously described in P and L groups was performed in group P+L. Venous blood sam?ples were taken at 120 min of reperfusion for determination of serum troponin I(TnI)and creatine kinase?MB(CK?MB)concentrations, myocardial infarct size(IS)and expression of phosphorylated GSK?3β [p?GSK?3β(Ser536)], NF?κBp65 and phosphorylated nuclear factor?κBp65(p?NF?κBp65)in myocar?dial tissues(by Western blot). Results Compared with group I∕R, myocardial IS and serum cTnI and CK?MB concentrations were significantly decreased, the expression of p?GSK?3β(Ser9)in ischemic area was up?regulated, and the expression of p?NF?κBp65 in ischemic area was down?regulated in P, L and P+L groups(P<0.05). Compared with group L, myocardial IS and serum cTnI and CK?MB concentrations were significantly decreased, the expression of p?GSK?3β(Ser9)in ischemic area was up?regulated, and the expression of p?NF?κBp65 in ischemic area was down?regulated in group P+L(P<0.05). Conclusion The mechanism by which α7nAChR agonist postconditioning alone or in combination with remote limb is?chemic postconditioning inhibits inflammatory responses during myocardial I∕R may be related to inhibiting GSK?3β activity in rats.
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Objective To evaluate the relationship between autophagy and diabetes mellitus-caused influence on ischemic preconditioning ( IP )-induced cardioprotection in rats. Methods Clean-grade healthy male Sprague-Dawley rats, aged 12 weeks, weighing 290-320 g, were used in this study. Diabe-tes mellitus was induced by high-fat and high-sucrose diet ( lasting for 1 week) and intraperitoneal streptozo-tocin 50 mg∕kg ( for 2 consecutive days) and confirmed by fasting blood glucose level≥16. 65 mmol∕L ( for 1 week) . Thirty rats with diabetes mellitus, weighing 350-450 g, were divided into 3 groups ( n=10 each) using a random number table method: sham operation group ( DM-S group) , myocardial ischemia-reperfusion ( I∕R) group ( DM-IR group) and IP group ( DM-IP group) . Another 30 non-diabetic rats were selected and divided into 3 groups ( n=10 each ) using a random number table method: sham operation group (S group), myocardial I∕R group (IR group) and IP group. Myocardial ischemia was induced by ligation of the anterior descending branch of left coronary artery for 30 min followed by 120 min reperfusion. IP was produced by 3 cycles of 5-min ischemia followed by 5-min reperfusion prior to establishment of myo-cardial I∕R injury model in IP and DM-IP groups. Blood samples were collected from the internal jugular vein at the end of reperfusion for measuring serum concentrations of cardiac troponin I ( cTnI) and creatine kinase-MB ( CK-MB) . The rats were then sacrificed and myocardial tissues were obtained for determination of myocardial infarct size and expression of microtubule-associated protein 1 light chain 3 Ⅱ ( LC3 Ⅱ) , Beclin-1, phosphatidyl-inositol 3-kinase (PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt) and mammalian target of rapamycin ( mTOR) ( by Western blot) . p-Akt∕Akt ratio was calculated. Results Compared with S group, the serum cTnI and CK-MB concentrations were significantly increased, the percentage of myocardial infarct size was increased, the expression of LC3Ⅱand Beclin-1 in myocardial tis-sues was up-regulated, the expression of PI3K and mTOR was down-regulated, and p-Akt∕Akt ratio was decreased in IR group (P<0. 05). Compared with IR group, the serum cTnI and CK-MB concentrations were significantly decreased, the percentage of myocardial infarct size was decreased, the expression of LC3Ⅱand Beclin-1 in myocardial tissues was down-regulated, the expression of PI3K and mTOR was up-regulated, and p-Akt∕Akt ratio was increased in IP group ( P<0. 05) . Compared with DM-S group, the se-rum cTnI and CK-MB concentrations were significantly increased, the percentage of myocardial infarct size was increased, the expression of LC3Ⅱ and Beclin-1 in myocardial tissues was up-regulated, the expres-sion of PI3K and mTOR was down-regulated, and p-Akt∕Akt ratio was decreased in DM-IR group ( P<0. 05) . There was no significant difference in the parameters mentioned above between DM-IP group and DM-IR group (P>0. 05). Conclusion The mechanism by which diabetes mellitus abolishes IP-induced cardioprotection may be related to inhibiting activation of PI3K-Akt-mTOR signaling pathway and enhanced autophagy in rats.
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<p><b>Background</b>Oxygen-glucose deprivation-nutrition resumption (OGD-NR) models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury (MIRI), but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI.</p><p><b>Methods</b>By searching the PubMed database using the keywords "myocardial reperfusion injury H9c2 cells," we obtained six commonly used OGD-NR in vitro models of MIRI performed on H9c2 cells from more than 400 published papers before January 30, 2017. For each model, control (C), simulated ischemia (SI), and simulated ischemia-reperfusion (SIR) groups were assigned, and cell morphology, lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory cytokines were examined to evaluate the characteristics of cell injury. Subsequently, a coculture system of cardiomyocyte-endothelial-macrophage was constructed. The coculture system was dealt with SI and SIR treatments to test the effect on cardiomyocytes survival.</p><p><b>Results</b>For models 1, 2, 3, 4, 5, and 6, SI treatment caused morphological damage to cells, and subsequent SIR treatment did not cause further morphological damage. In the models 1, 2, 3, 4, 5 and 6, LDH release was significantly higher in the SI groups than that in the C group (P < 0.05), and was significantly lower in the SIR groups than that in the SI groups (P < 0.05), except for no significant differences in the LDH release between C, SI and SIR groups in model 6 receiving a 3-h SI treatment. In models 1, 2, 3, 4, 5, and 6, compared with the C group, ATP levels of the SI groups significantly decreased (P < 0.05), ROS levels increased (P < 0.05), and MMP levels decreased (P < 0.05). Compared with the SI group, ATP level of the SIR groups was significantly increased (P < 0.05), and there was no significant ROS production, MMP collapse, and over inflammatory response in the SIR groups. In a coculture system of H9c2 cells-endothelial cells-macrophages, the proportion of viable H9c2 cells in the SIR groups was not reduced compared with the SI groups.</p><p><b>Conclusion</b>All the six OGD-NR models on H9c2 cells in this experiment can not imitate the characteristics of MIRI in vivo and are not suitable for MIRI-related study.</p>
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Humans , Apoptosis , Glucose , Metabolism , Myocardial Reperfusion Injury , Myocytes, Cardiac , Physiology , Oxygen , MetabolismABSTRACT
<p><b>Background</b>Ischemia preconditioning (IPC) remains the most powerful intervention of protection against myocardial ischemia/reperfusion injury (IRI), but diabetes can weaken or eliminate its cardioprotective effect and detailed mechanisms remain unclear. In this study, we aimed to explore whether changes of autophagy in the diabetic condition are attributable to the decreased cardioprotective effect of IPC.</p><p><b>Methods</b>Sixty diabetic male Sprague-Dawley rats were randomly divided into the control (C), IRI, rapamycin (R), wortmannin (W), rapamycin + IPC (R + IPC), and wortmannin + IPC (W + IPC) groups. The in vivo rat model of myocardial IRI was established by ligaturing and opening the left anterior descending coronary artery via the left thoracotomy. Durations of ischemia and reperfusion are 30 min and 120 min, respectively. Blood samples were taken at 120 min of reperfusion for measuring serum concentrations of troponin I (TnI) and creatine kinase isoenzyme MB (CK-MB) using the enzyme-linked immunosorbent assay. The infarct size was assessed by Evans blue and triphenyltetrazolium chloride staining. The expressions of LC3-II, beclin-1, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), and P-Akt/Akt ratio in the ischemic myocardium were assessed by Western blotting.</p><p><b>Results</b>Compared to the IRI group, infarct size (56.1% ± 6.1% vs. 75.4 ± 7.1%, P < 0.05), serum cTnI (0.61 ± 0.21 vs. 0.95 ± 0.26 ng/ml, P < 0.05), and CK-MB levels (6.70 ± 1.25 vs. 11.51 ± 2.35 ng/ml, P < 0.05) obviously decreased in the W + IPC group. Compared with the C group, myocardial expressions of LC3-II (0.46 ± 0.04 and 0.56 ± 0.04 vs. 0.36 ± 0.04, P < 0.05) and beclin-1 (0.34 ± 0.08 and 0.38 ± 0.07 vs. 0.24 ± 0.03, P < 0.05) evidently increased, and myocardial expressions of mTOR (0.26 ± 0.08 and 0.25 ± 0.07 vs. 0.38 ± 0.06, P < 0.05), PI3K (0.29 ± 0.04 and 0.30 ± 0.03 vs. 0.38 ± 0.02, P < 0.05), and P-Akt/Akt ratio (0.49 ± 0.10 and 0.48 ± 0.06 vs. 0.72 ± 0.07, P < 0.05) markedly decreased in the IRI and R groups, indicating an increased autophagy. Compared with the IRI group, myocardial expression of beclin-1 (0.26 ± 0.03 vs. 0.34 ± 0.08, P < 0.05) significantly decreased, and myocardial expressions of mTOR (0.36 ± 0.04 vs. 0.26 ± 0.08, P < 0.05), PI3K (0.37 ± 0.03 vs. 0.29 ± 0.04, P < 0.05), and P-Akt/Akt ratio (0.68 ± 0.05 vs. 0.49 ± 0.10, P < 0.05) increased obviously in the W + IPC group, indicating a decreased autophagy.</p><p><b>Conclusions</b>Increased autophagy in the diabetic myocardium is attributable to decreased cardioprotection of IPC, and autophagy inhibited by activating the PI3K-Akt-mTOR signaling pathway can result in an improved protection of IPC against diabetic myocardial IRI.</p>
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Objective To evaluate the role of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)or Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT-3)signaling pathways in reduction of myocardial ischemia-reperfusion(I/R)injury by combination of limb ischemic and morphine postconditioning in rats.Methods Eighty SPF healthy male Sprague-Dawley rats,aged 8 weeks,weighing 280-320 g,were used in the study.Myocardial I/R was induced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.The rats were divided into 8 groups(n=10 each)using a random number table:I/R group,limb ischemic postconditioning group(LIP group),morphine postconditioning group(group MP),combination of limb ischemic and morphine postconditioning group(LIP+MP group)and signaling pathway blocker groups(I/Rb group,LIPb group,MPb group,LIP+MPb group).In I/R,LIP,MP and LIP+MP groups,the animals were sacrificed at the end of reperfusion,and myocardial specimens in ischemic and non-ischemic regions were obtained for determination of phosphorylated STAT-3(p-STAT-3),STAT-3,phosphorylated Akt(p-Akt)and Akt expression(by Western blot)and STAT-3 and Akt mRNA expression(by polymerase chain reaction).In I/Rb,LIPb,MPb and LIP+MPb groups,PI3K/Akt signaling pathway blocker LY294002 0.3 mg/kg was intravenously injected in 5 rats of each group,and JAK/STAT-3 signaling pathway blocker AG490 5 mg/kg was intravenously injected in the other 5 rats of each group.The animals were sacrificed at the end of reperfusion,and myocardial specimens in the ischemic region were obtained for determination of myocardial infarct size.Results Compared with I/R group,the p-STAT-3/STAT-3 ratio in LIP,MP and LIP+MP groups and p-Akt/Akt ratio in LIP+MP group were significantly increased,and the expression of STAT-3 and Akt mRNA was up-regulated in LIP+MP group(P0.05).When JAK2 inhibitor AG490 was applied,the myocardial infarct size was significantly smaller in LIP+MPb group than in I/Rb,LIPb and MPb groups(P0.05).Conclusion Combination of limb ischemic and morphine postconditioning can enhance the activation of PI3K/Akt or JAK/STAT-3 signaling pathways,and the cardioprotection is dependent on the integrity of the PI3K/Akt signaling pathway and partially dependent on the integrity of the JAK/STAT-3 signaling pathway when applied in combination in rats.
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<p><b>BACKGROUND</b>Both ischemic preconditioning (IPC) and limb remote ischemic postconditioning (LRIPOC) have been shown to possess significantly different cardioprotective effects against the myocardial ischemia reperfusion injury (IRI), but no study has compared the anti-inflammatory effects of IPC and LRIPOC during myocardial IRI process. We hypothesized that IPC and LRIPOC would produce different anti-inflammatory effects in an in vivo rat model with myocardial IRI.</p><p><b>METHODS</b>Eighty rats were randomly allocated into four equal groups: sham group, IRI group, IPC group and LRIPOC group. In 10 rats randomly selected from each group, serum levels of TNF-α, HMGB1, ICAM1, IL-1, IL-6 and IL-10 were assessed, and infarct size was determined. In another 10 rats of each group, myocardial levels of TNF-α, HMGB1, ICAM1, IL-1, IL-6 and IL-10 in both ischemic and non-ischemic regions were measured.</p><p><b>RESULTS</b>The infarct size was significantly lower in IPC and LRIPOC groups than in IRI group. The serum and myocardial levels of pro-inflammatory cytokines including TNF-α, HMGB1, ICAM1, IL-1 and IL-6 during reperfusion were significantly reduced in IPC and LRIPOC groups compared to IRI group. As compared to the IPC group, infarct size, serum level of TNF-α at 60 minutes of reperfusion, serum levels of HMGB1 and ICAM1 at 120 minutes of reperfusion, myocardial levels of TNF-α, ICAM1, IL-1 and IL-6 in the ischemic region, myocardial levels of ICAM1, IL-1 and IL-6 in the non-ischemic region were significantly increased in the LRIPOC group.</p><p><b>CONCLUSIONS</b>In the rats with myocardial IRI, IPC produces more powerful inhibitory effects on local myocardial and systemic inflammatory responses than LRIPOC. This may be partly attributed to more potent cardioprotection produced by IPC.</p>
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Animals , Male , Rats , HMGB1 Protein , Metabolism , Intercellular Adhesion Molecule-1 , Metabolism , Interleukin-1 , Metabolism , Interleukin-10 , Metabolism , Interleukin-6 , Metabolism , Ischemic Postconditioning , Methods , Ischemic Preconditioning , Methods , Myocardial Reperfusion Injury , Allergy and Immunology , Metabolism , Therapeutics , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha , MetabolismABSTRACT
<p><b>BACKGROUND</b>Inflammation is one of important mechanisms for myocardial ischemia reperfusion injury (IRI). Ischemia postconditioning (IPOC) can protect the heart against IRI by inhibiting inflammation, but its cardioprotection is weaker than that of ischemia preconditioning. Recently, the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist has shown anti-inflammatory effects in many diseases related to inflammation. This randomized controlled experiment was designed to evaluate whether combined postconditioning with IPOC and the α7nAChR agonist could produce an enhanced cardioprotection in a rat in vivo model of acute myocardial IRI.</p><p><b>METHODS</b>Fifty Sprague-Dawley rats were randomly divided into five equal groups: sham group, control group, IPOC group, α7nAChR agonist postconditioning group (APOC group) and combined postconditioning with IPOC and α7nAChR agonist group (combined group). Hemodynamic parameters were recorded during the periods of ischemia and reperfusion. Serum concentrations of troponin I (TnI), tumor necrosis factor α (TNF-α) and high-mobility group box 1 (HMGB-1) at 180 minutes after reperfusion were assayed in all groups. At the end of the experiment, the infarct size was assessed from excised hearts by Evans blue and triphenyl tetrazolium chloride staining.</p><p><b>RESULTS</b>As compared to the sham group, the infarct size in the other four groups was significantly increased, serum levels of TnI, TNF-α and HMGB1 in the control group and TNF-α, HMGB1 in the IPOC group were significantly increased. The infarct size and serum concentrations of TNF-α, HMGB1 and TnI in the IPOC, APOC and combined groups were significantly lower than those in the control group. As compared to the IPOC group, the infarct size in the combined group was significantly decreased, serum concentrations of TnI, TNF-α and HMGB1 in the APOC and combined groups were significantly reduced. Although the infarct size was significantly smaller in the combined group than in the APOC group, serum levels of TNF-α and HMGB1 were significantly higher in the combined group than in the APOC group.</p><p><b>CONCLUSIONS</b>In a rat in vivo model of acute myocardial IRI, combined postconditioning with IPOC and the α7nAChR agonist can produce enhanced protection against myocardial IRI by increasing the anti-inflammatory effect.</p>
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Animals , Male , Rats , Heart , Ischemic Preconditioning, Myocardial , Methods , Myocardial Reperfusion Injury , Myocardium , Pathology , Nicotinic Agonists , Therapeutic Uses , Rats, Sprague-Dawley , Receptors, Nicotinic , Metabolism , Tumor Necrosis Factor-alpha , Blood , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
Objective To investigate the role of reactive oxygen species (ROS) in the reduction of myocardial ischemia-reperfusion (I/R) injury by fentanyl postconditioning and remote limb ischemic postconditioning in rats.Methods Sixty-three male Sprague-Dawley rats,aged 8 weeks,weighing 250-350 g,were equally and randomly allocated into 7 groups:sham operation group (group S),group I/R,fentanyl postconditioning group (group F),remote limb ischemic postconditioning group (group R),ROS scavenger N-(2-Mercaptopropionyl) glycine (MPG) group (group M),MPG + fentanyl postconditioning group (group MF),and MPG + remote limb ischemic postconditioning group (group MR).Myocardial I/R was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 180 min of reperfusion.In group S the anterior descending branch was only exposed but not ligated.MPG 5 mg/kg was infused intravenously from 5 min before ischemia to 15 min of reperfusion in groups M,MF and MR,while the equal volume of normal saline was given in the other four groups.In groups F and MF,fentanyl 30 μg/kg was injected intravenously at 15 min of myocardial ischemia.In groups R and MR,the animals underwent 10 min ischemia of bilateral hind limbs starting from 15 min of myocardial ischemia.Arterial blood samples were taken at 180 min of reperfusion to determine the serum cardiac troponin I (cTnI) concentration.The rats were then sacrificed.The infarct size was measured by TTC.Results Compared with group S,the serum cTnI concentration and infarct size were significantly increased in the other six groups (P ＜0.05).Compared with group I/R,no significant change was found in the serum cTnI concentration and infarct size in M group,and the serum cTnI concentration and infarct size were significantly decreased in F and R groups (P ＜ 0.05).There was no significant difference in the serum cTnI concentration and infarct size between MF group and F group (P ＞ 0.05).The serum cTnI concentration was significantly higher and the infarct size was larger in group MR than in group R (P ＜ 0.05).Conclusion ROS is involved in the reduction of myocardial I/R injury by remote limb ischemic postconditioning in rats,but not in the myocardial protection provided by fentanyl postconditioning.
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Objective To evaluate the roles of PI3K/Akt and JAK/STAT signal transduction pathways in reduction of myocardial ischemia/reperfusion (I/R) injury by postconditioning with α subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist in rats.Methods Sixty Sprague-Dawley rats,weighing 290-320 g,were randomly divided into 4 groups (n =15 each):I/R group,ischemic preconditioning group (IPC group),ischemic postconditioning group (IPOC group) and postconditioning with specific α7nAChR agonist PNU282987 group ( PNU group ).Myocardial I/R was produced by 30 min occlusion of left anterior descending coronary artery followed by 180 min reperfusion in the 4 groups.The animals were subjected to 3 cycles of 5 min myocardial ischemia and 5 min reperfusion before 30 min myocardial ischemia in IPC group.The animals underwent 3 cycles of 10 s myocardial ischemia at 5 s intervals before 180 min reperfusion in group IPOC.PNU282987 2.4 mg/kg was injected intraperitoneally immediately before the reperfusion.At 60 min of reperfusion,5 rats in each group were sacrificed and the hearts were removed to determine the expression of Akt and STAT3 mRNA,phosphorylated Akt (p-Akt) and phosphorylated STAT3 (p-STAT3) in myocardial tissues.The left 10 rats in each group were sacrificed at 180 min of reperfusion and the hearts were removed to measure the infarct size.Results Compared with I/R group,the expression of STAT3 mRNA and p-Akt was significantly up-regulated in IPC group,and the expression of p-Akt and p-STAT3 was significantly up-regulated in IPOC group ( P ＜ 0.05).The infarct size was significantly reduced in IPC,IPOC and PNU groups compared with I/R group ( P ＜ 0.05 ).Conclusion The mechanism by which α7nAChR agonist postconditioning reduces myocardial I/R injury is not related to PI3K/Akt and JAK/STAT signal transduction pathways in rats.
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The selection of pre-embryos for transferred is based on morphological appearance. But some poor quality cleaved embryos also can be cultured to the blastocyst stage and implanted. To assess the clinical pregnancy outcomes of blastocyst transfer which developed from poor quality embryos. A total of 109 cleaved embryos with poor quality were cultured to day 5/day 6 and 27 [24.8%] blastocysts were collected from the 15 cycles/patients undergoing conventional IVF. All the blastocysts were cooling with fast-freezing. Then the blastocysts were warmed for transfer. All of 25 vitrified blastocysts [92.6%] survived after warming and were transferred to 15 patients. Five of the women became pregnant. Our results suggest that vitrified human day 5/day 6 blastocyst transfer which develop from poor quality embryo at day 3 can contribute to increasing cumulative pregnancy rates in assisted reproduction
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<p><b>OBJECTIVE</b>A general review was made of studies involving: (1) The experimental evidence of remote ischemic preconditioning (RIPC) and relative clinical studies, (2) The experimental and clinical evidences of remote ischemic postconditioning (RIPOC), (3) The potential mechanistic pathways underlying their protective effects.</p><p><b>DATA SOURCES</b>The data used in this review were mainly from manuscripts listed in PubMed that were published in English from 1986 to 2010. The search terms were "myocardial ischemia reperfusion injury", "ischemia preconditioning", "ischemia postconditioning", "remote preconditioning" and "remote postconditioning".</p><p><b>STUDY SELECTION</b>(1) Clinical and experimental evidence that both RIPC and RIPOC produce preservation of ischemia reperfusion injury (IRI) of myocardium and other organs, (2) Studies related to the potential mechanisms, by which remote ischemic conditioning protects myocardium against IRI.</p><p><b>RESULTS</b>Both RIPC and RIOPC have been shown to attenuate myocardial IRI in laboratory animals. Also, their cardioprotective effects have appeared in some clinical studies. Except the external, the detailed internal mechanisms of remote ischemic conditioning have been generally described. Through these descriptions better protocols can be developed to provide improved cardioprotective procedures.</p><p><b>CONCLUSIONS</b>Remote ischemic conditioning is an endogenous cardioprotective mechanism from outside the heart that protects against myocardial IRI and represents a general form of inter-organ protection. Remote ischemic conditioning may have an immense impact on clinical practice in the near future.</p>
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Humans , Ischemic Preconditioning, Myocardial , Methods , Myocardial Reperfusion InjuryABSTRACT
ObjectiveTo evaluate the effects of combined vagus nerve electric stimulation postconditioning and limb remote ischemic postconditioning on myocardial ischemia-reperfusion (I/R) injury in rats.Methods One hundred male SD rats aged 8 weeks weighing 250-350 g were randomly allocated into 5 groups ( n =20 each):sham operation group (group S); I/R group; vagus nerve electric stimulation postconditioning group (group POES) ; limb remote ischemic postconditioning group (group RP) and vagus nerve electric stimulation postconditioning + limb remote ischemic postconditioning group (group POES-RP).Myocardial I/R was induced by occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 120 min reperfusion in groups I/R,POES,RP and POES-RP.In groups POES and POES-RP,right cervical vagus nerve trank was stimulated for 30 min with continuous electric rectangular pulses (2 ms,10 Hz) starting from 15 min of myocardial ischemia.The voltage of the pulses was adjusted to decrease HR by 10％ of the baseline HR before stimulation.In groups RP and POES-RP the animals underwent 10 min ischemia of bilateral hind limbs starting from 20 min of myocardial ischemia.Arterial blood samples were collected from 10 rats in each group at 120 min of reperfusion for determination of serum concentrations of cTnI,CK-MB,TNF-α,high mobility group box 1 protein (HMGB1),intercellular adhesion molecule-1(ICAM-1),IL-1,IL-6 and IL-10 (by ELISA).The animals were then sacrificed and myocardial infarct size was measured by Evans blue and TTC staining.Another 10 rats were sacrificed at 120 min of reperfusion for determination of myocardial contents of TNF-α,HMGB-1,ICAM-1,IL-1,ID6 and IL-10 (by ELISA).ResultsI/R induced myocardial infarct and significantly increased serum concentrations of cTnI,CK-MB,TNF-α,HMGBi,ICAM-1,IL-1 and IL-6 and increased myocardial contents ofTNF-α,HMGB1,ICAM-1,IL-1,IL-6 and IL-10 in both ischemic and non-ischemic regions in group I/R as compared with group S.Vagus nerve electric stimulation postconditioning,limb remote ischemic postconditioning and vagus nerve electric stimulation postconditioning + limb remote ischemic postconditioning significantly decreased myocardial infarct size and serum concentrations of cTnI,CK-MB,TNF-α,HMGB1,ICAM-1,IL-1 and IL-6 and decreased myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1,IL-6 in groups POES,RP and POES-RP as compared with group I/R.Compared with group I/R,myocardial IL-10 content in both ischemic and non-ischemic regions was significantly increased in groups POES and POES-RP.Compmared with group POES,myocardial infarct size,serum concentrations of cTnI,CK-MB,TNF-α,ICAM-1 and myocardial contents of ICAM-1 and IL-6 in ischemic region were significantly decreased,while myocardial content of IL-10 in non-ischemic region was increased in group POES-RP.Compared with group RP,myocardial infarct size,serum concenuations of cTnI,CK-MB,TNF-α,HMGB1,ICAM-1,IL-1,IL-6 and myocardial contents of TNF-α,ICAM-1,IL-1 and IL-6 in ischemic region were significantly decreased,myocardial content of IL-10 in ischemic region was increased and HMGB1,ICAM-1,IL-1 and IL-6 contents were decreased,IL-10 content was increased in myocardial of ischemic region in group POES-RP.ConclusionMyocardial I/R injury is attenuated and myocardial protection is enhanced by combination of vagus nerve electric stimulation postconditioning and limb remote ischemic postconditioning in rats by inhibiting inflammatory response in rats.
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Objective To investigate the effects of preconditioning with different doses of levothyroxine sodium on myocardial ischemia-reperfusion (I/R) injury in immature rats. Methods Forty-eight female immature Wistar rats, aged 35 days, weighing 120-140 g, were randomly allocated into 6 groups ( n = 8 each): control group (group C), I/R group, and preconditioning with levothyroxine sodium 10, 20, 40 and 80 μg/100 g groups (groups LS1-4 ) . The rats received levothyroxine sodium 10, 20, 40 and 80 μg/100 g through a gastric tube every day for 7 days in groups LS1-4 , respectively. Venous blood samples were taken on 8th day for determination of serum thyroid hormone levels. The hearts were removed from the animals and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃. The hearts were continuously perfused for 80 min in group C. After 30 min of equilibration, the isolated hearts were subjected to 20 min of ischemia followed by 30 min of reperfusion in I/R and LS1-4 groups. HR, SP and ± dp/dtmax were recorded at 20 min of perfusion and 30 min of reperfusion. The recovery rates of HR, SP and ± dp/dtmax were calculated at 30 min of reperfusion. The coronary effluent was collected at 10 min of perfusion and 15 min of reperfusion for determination of creatine kinase (CKMB) activity. The samples of ventricular myocardial tissues were taken at 30 min of reperfusion to detect the expression of heat shock protein 70 (HSP70), thyroid hormone receptor (TR) mRNA (TRa, , TRoj and TRft ) and myosin heavy chain (MHC) mRNA. Results Compared with group C, the recovery rates of HR, SP and. ± dp/dtmax were significantly decreased, the CK-MB activity was significantly increased, and MHCα mRNA expression was down-regulated in group I/R, the recovery rates of SP and ± dp/dtmax were significantly decreased, the CK-MB activity was significantly increased, and the expression of HSP70 and MHCα mRNA was up-regulated in groups LS1-4, and the serum thyroid hormone level was significantly increased and the expression of TRa, mRNA was up-regulated in groups LS2-4 ( P ＜ 0.05) . Compared with group I/R, the recovery rates of HR and ± dp/dtmax were significantly increased, the pression of HSP70 and MHCa mRNA was up-regulated, and the MHCJ3 mRNA expression was down-regulated in groups LS1-4 the CK-MB activity was significantly decreased in groups LS1-3, and the serum thyroid hormone level was significantly increased and the expression of TRα1, mRNA was up-regulated in groups LS2-4 ( P ＜ 0.05) . The serum thyroid hormone level increased gradually with the increase in the dose of levothyroxine sodium in groups LS1-4 ( P ＜ 0.05) . The CK-MB activity was significantly higher, while the HSP70 expression lower in groups LS3-4 than in groups LS1-2 (P ＜ 0.05). Conclusion Preconditioning with levothyroxine sodium 10 μg/100 g can alleviate the myocardial I/R injury in immature rats and does not lead to the increase in the level of thyroid hormone, and the up-regulation of HSP70 and MHCa mRNA expression may be involved in the mechanism.
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ObjectiveTo investigate the effects of postconditioning with electric vagal stimulation on myocardial ischemia-reperfusion (I/R) injury in rats.MethodsSixty male SD rats weighing 250-350 g were randomly divided into 3 groups (n ＝ 20 each):group sham operation (group S); group myocardial I/R (group I/R) and group electric vagal stimulation postconditioning (group POES).Myocardial I/R was induced by occlusion of left anterior descending branch of coronary artery for 30 min followed by 120 min reperfusion in groups I/R and POES.In group POES right cervical vagus nerve trunk was stimulated for 30 min with continuous electric rectangular pulses (2 ms,10 Hz) starting from 15 min of myocardial ischemia.The voltage of the pulses was adjusted to decrease HR by 10％ of the baseline HR before stimulation.MAP,HR and RPP (MAP× HR) were recorded before (baseline) and at 1 and 10 min of ischemia and 30,60 and 120 min of reperfusion.Arterial blood samples were collected from 10 rats in each group at 120 min of reperfusion for determination of serum concentrations of cTnI,CK-MB,TNF-a,high mobility group box 1 protein (HMGB1),ICAM-1,IL-1,IL-6 and IL-10 (by ELISA).The animals were then sacrificed and myocardial infarct size was measured by Evans blue and TTC staining.Another 10 rats were sacrificed at 120 min of reperfusion for determination of myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1,IL-6 and IL-10 (by ELISA).ResultsI/R induced myocardial infarct,significantly increased serum concentrations of cTnI,CK-MB,TNF-α,HMGB1,ICAM-1,IL-1 and IL-6 and significantly increased myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1,IL-6 and IL-10 in both ischemic and non-ischemic regions in group I/R as compared with group S.Electric vagal stimulation significantly decreased myocardial infarct size and serum concentrations of cTnI,CK-MB,TNF-α,HMGB1,1CAM-1,IL-1 and IL-6 in group POES compared with group I/R.Myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1 and IL-6 were significantly decreased while myocardial IL-10 content was increased in both ischemic and non-isehemic regions in groups POES compared with group I/R.ConclusionPostconditioning with electric vagal stimulation can attenuate myocardial I/R injury by inhibiting inflammatory response in rats.
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Objective To compare the effects of ischemic preconditioning versus ischemic postconditioning on myocardial ischemia-reperfusion (I/R)-induced inflammatory response in rats. Methods Forty male SD rats weighing 290-320 g were randomly divided into 4 groups ( n = 10 each): Ⅰ group sham operatin (group S); Ⅱ group I/R; Ⅲ group ischemic preconditioning (group IPC) and Ⅳ group ischemic postconditioning (group IPOC).Myocardial I/R was induced by 30 min ligation of left anterior descending branch (LAD) of coronary artery followed by reperfusion. In group IPC myocardial I/R was preceded by 3 cycles of ischemia followed by reperfusion (each lasting 5 min) while in group IPOC 3 cycles of I/R (each lasting 10 s) was started at the end of 30 min myocardial ischemia. MAP, HR and RPP ( MAP × HR) were recorded before (baseline) and at 1 and 20 min of ischemia and 60, 120 and 180 min of reperfusion. Venous blood samples were collected at 30 and 180 min of reperfusion for determination of serum concentrations of TNF-α, IL-6, high-mobility group box 1 (HMGB1) and cTnI. The animals were sacrificed at 180 min of reperfusion and the myocardial infarct size was measured. Results Myocardial I/R significantly decreased MAP and RPP and increased myocarcial infarct size, serum concentrations of TNF-α, IL-6,HMGB1 and cTnI in group I/R as compmed with group S. Ischemic pre- and postconditioning significantly increased MAP and reduced myocardial infarct size and I/R-induced increase in serum TNF-α, HMGB1 and cTnI concentrations in group Ⅲ and Ⅳ as compared with group Ⅱ (I/R). The myocardial infarct size was significantly larger and the serum concentrations of TNF-α, IL-6 and HMGB1 were significantly higher in ischemic postconditioning group than in the preconditioning group. Conclusion Ischemic preconditioning is more effective in attenuating the myocardial I/R-induced inflammatory response than the ischemic postconditioning.
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Objective To investigate whether naloxone postconditioning could attenuate the focal cerebral ischemia-reperfusion (I/R) injury in rats. Methods Eighty-eight adult male SD nits weighing 270-330 g were randomly divided into 4 groups (n = 22 each) : group I sham operation (S); group Ⅱ I/R; group Ⅲ , Ⅳ I/R + low and high dose naloxone ( N_1, N_2). Focal cerebral I/R was produced by occlusion of right middle cerebral artery for 90 min followed by 24 h reperfusion. In group N_1, and N_2 naloxone 1 and 10 mg/kg were injected intraperitoneally at initiation of reperfusion respectively. In group I/R normal saline was injected instead of naloxone. HR, MAP and EKG were continuously monitored throughout the experiment. He neurological deficits were scored (0 = no deficit, 4 = unable to crawl, mental dysfunction) at 2 h and 24 h of reperfusion. The animals were then decapitated. The brains were immediately removed for determination of infarct size ( n = 10) and the expression of microtubule-associated protein-2 ( MAP-2) in brain tissue ( n = 6) . In the other 6 rats in each group FICT-dextran 1 ml (50 mg/ml) was injected iv at 1 min before decapitation. The cerebral plasma volume and diameter and segment length of cerebral microvessels on the I/R side were measured using laser scanning confocal microscopy (LSCM). Results Focal cerebral I/R significantly increased neurological deficit scores, induced cerebral infarct, and decreased MAP-2 expression in the brain tissue, cerebral plasma volume and the diameter and segment length of cerebral microvessels on the I/R side. Postconditioning with 10 mg/kg naloxone significantly attenuated the above-mentioned focal cerebral I/R-induced changes. Conclusion Postconditioning with naloxone can attenuate focal cerebral I/R injury in a dose-dependent manner.
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Objective To investigate the role of cyclooxygenase-2(COX-2)and mitochondrial adenosine tuiphosphate sensitive potassium channels (mito-KATP channels) in sufentanil preconditioning-induced delayed cardiopreteetion against myocardial ischemia-reperfnsion (I/R) injury in rats. Methods Seventy-two adult male Wistsr rats weighing 250-300 g were randomly divided into 6 groups ( n =12 each). Group Ⅰ,Ⅱ,Ⅲ were preconditioned with intraperitoneal (IP) normal saline (NS) 1 ml/kg while group Ⅳ,Ⅴ,Ⅵ with IP sufentanil 20 μg/kg at 24 h before myocardial ischemia. Group Ⅱ and Ⅴ were given IP NS-398 ( COX-2 inhibitor) 5 mg/kg at 30 rain before myocardial ischemla while group Ⅲ and Ⅵ were given intravenous 5-HD (mito-KATP channelblocker) 10 mg/kg at 10 min before ischemia or before being killed. Six animals in each group underwent 45 min myocardial ischemia followed by 120 min reperfusion, while the other six animals in each group were killed immediately before ischemia for determination of myocardial COX-2 expression and myocardial PGF2 and PGF1α content. Myocardial ischemia was induced by occlusion of left anterior descending branch (LAD) of coronary artety for 45 rain followed by 120 min reperfusion. MAP and HR were recorded immediately before ischemia (T0), at 15, 30, 45 rain of ischemia (T1-3) and at 30, 60, 90, 120 vain of reperfusion (T4-7). Heart rate-blood pressure product (RPP) was calculated. Arterial blood samples were obtained at T0.3 and T7 for measurement of plasma CK-MB activity. The animals were killed at the end of 120 nan reperfusion. The hearts were removed for determination of myocardial infarct area (IA) and area at risk (AAR). LA/AAR was calculated. Results There was no significant difference in HR, MAP and RPP at all time points among the 6 groups. Preconditioning with sufentanil significantly decreased plasma CK-MB activity at T3 and T7 and IA/AAR in group Ⅳ as compared with group Ⅰ.Myocardial COX-2 expression was up-regulated and PGE2 and PGF1α, contents were elevated by sufentanil preconditioning in group Ⅳ as eomared with control group (Ⅰ). In group Ⅴ and Ⅳ preconditioning with NS-398/5-HD significantly increased plasma CK-MB concentration and IS/AAB as compared with group Ⅳ, indicating involvement of COX-2 and mito-KATP channels in the sufentanil-induced delayed cardioprotection.The myocardial PGE2 and PGF1α contents were significantly reduced in group Ⅴ as compared with group Ⅳ. There was no significant difference in the myocardial COX-2 expression among group Ⅳ, Ⅴ and Ⅵ. Conclusion Both COX-2 and mito-KATP channels are involved in sufentanil preconditioning-induced delayed cardiopmtection.
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Objective To evaluate the protective effects of fentanyl postconditioning and remote limb ischemic postconditioning (RLIP) against myocardial ischemia-reperfusion (I/R) injury in rats. Methods Thirty-nine male SD rats aged 8 weeks weighing 250-350 g were randomly allocated into 5 groups: Ⅰ group sham operation (group S, n = 5); Ⅱ group I/R ( n = 7); Ⅲ group fentanyl postconditioning (group F, n= 9); Ⅳ group RLIP (group R, n = 9) and group Ⅴ fentanyl postconditioning + RLIP (group F-R, n = 9). The animals were anesthetized with intraperitoneal 3% pentobarbital 50 mg/kg, intubated and mechanically ventilated. Myocardial I/R was induced by occlusion of anterior desending branch of left coronary artery for 30 min followed by 180 min reperfusion. Fentanyl 30 μg/kg was injected iv at 15 min of myocardial ischemia in group F and F-R In group R and F-R the animals underwent 10 min ischemia of bilateral hind limbs starting from 15 min of myocardial ischemia. HR and MAP were recorded at 5,60,120 and 180 min of reperfusion and rate-pressure product( RPP, HR × MAP) were calculated. At the end of 180 min reperfusion, arterial blood samples were obtained for measurement of the activities of plasma lactate dehydrogenase (LDH) and creatine kinase isoenzyme MB (CK-MB), and the concentration of serum cardiac troponin Ⅰ (cTnI). The animals were then sacrificed. The infarct size was evaluated by double staining with Evans blue and triphenyl tetrazolium chloride. Results Myocardial I/R significantly increased plasma LDH and CK-MB activities and serum cTnI concentration and decreased HR,MAP and RPP as compared with group S.Fentanyl postconditioning and RLIP both decreased plasma CK-MB activity, serum cTnI concentration and infarct size and increased HR, MAP and RPP in group F, R and F-R as compared with group I/R. Plasma CK-MB activity,serum cTnI concentration and RPP were significantly lower and infarct size was smaller in group F-R than in group F. The infarct size was significantly smaller and MAP and RPP were higher in group F-R than in group R.Conclusion Fentanyl postconditioning can provide a myocardial protection against I/R injury. Myocardial protection is enhanced by combination of fentanyl postconditioning and RLIP.
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Objective To investigate the effects of postconditioning with α7 nicotinic acetylcholine receptor (α7nAChR) agonist and ischemia on myocardial ischemia-reperfusion (IR) injury in rats. Methods Fifty adult male SD rats weighing 290-320 g were randomly divided into 5 groups ( n = 10 each): Ⅰ sham operation group, Ⅱ IR group, Ⅲ ischemic postconditioning group, Ⅳ α7nAChR agonist postconditioning group and Ⅴpostconditioning with α7nAChR agonist and ischemia group. Myocardial I/R was induced by ligation of anterior descending branch of left coronary artery for 30 min followed by 1 80 min of reperfusion. In group] the anterior descending branch was only exposed but not ligated. In group Ⅲ the hearts were subjected to 3 episodes of 10 second ischemia at 10 second intervals at the end of 30 min ischemia before 180 min reperfusion, Intraperitoneal PNU282987 2.4 mg/kg was injected at the end of 30 min ischemia before 180 min reperfusion in group Ⅳ and Ⅴ .Blood samples were taken from right internal jugular vein at 180 min of reperfusion. Then the rats were killed and hearts removed to determine the concentrations of serum cardiac troponin-I (cTnI), TNF-α and high-mobility group box 1 (HMGB1) by ELISA. The infarction size was measured by Evans blue and triphenyltetrazolium chloride staining. Results The infarction size was significantly larger in the other groups and concentrations of serum cTrI, TNF-α and HMGB1 were significantly higher in group Ⅱ than in group Ⅰ ( P ＜ 0.05). The infarction size was significantly smaller and concentrations of serum cTnI, TNF-α and HMGBI were significantly lower in group Ⅲ, Ⅴ than in group Ⅱ (P ＜ 0.05). The infarction size was significantly smaller in group Ⅴ and concentrations of serum cTnI, TNF-α and HMGB1 were significantly lower in group Ⅳ and Ⅴ than in group Ⅲ (P ＜0.05). The infarction size was significantly smaller and concentrations of serum cTnI, TNF-α and HMGB1 were significantly lower in group Ⅴ than in group Ⅳ ( P ＜ 0.05 ). Conclusion Postconditioning with α7nAChR agonist and ischemia can reduce myocardial I/R injury and the efficacy is better than that of α7nAChR agonist postconditioning or ischemic postconditioning alone.