Reduction of apoptosis in the amygdala by an A2A adenosine receptor agonist following myocardial infarction

It has been observed that a cytokine synthesis inhibitor, pentoxifylline, prevents the apoptotic processes taking place in the amygdala following myocardial infarction. However, it is unknown if the cardioprotective effect of A_2A adenosine receptor agonist, CGS21680, which reduces cytokine synthesis, would lead to such amygdala apoptosis regression. Thus, this study was designed to investigate whether cardioprotective A_2A adenosine receptor activation reduces apoptosis in the amygdala following myocardial infarction. Anesthetized rats were subjected to left anterior descending coronary artery occlusion for 40 min, followed by 72 h of reperfusion. The A_2A agonist CGS21680 (0.2 μg/kg/min i.v.) was administered continuously for 120 min, starting (1) five minutes prior to instituting reperfusion (Early) or (2) five minutes after the beginning of reperfusion (Late). After reperfusion, myocardial infarct size was determined and the amygdala was dissected from the brain. Infarct size was reduced significantly in the Early compared to the Control group (34.6 ± 1.8% and 52.3 ± 2.8% respectively; p < 0.05), with no difference com-pared to the Late group (40.1 ± 6.1%). Apoptosis regressi-on was documented in the amygdala of the Early group by an enhanced phosphatidylinositol 3-kinase-Akt pathway activation and Bcl-2 expression concurrently to a caspase-3 activation limitation and reduction in TUNEL-positive cells staining. On the other hand, amygdala TUNEL-positive cell numbers were not reduced in the Late group. Moreover, TNFα was significantly reduced in the amygdala of the Early group compared to the Control and Late groups. These results indicate that A_2A adenosine receptor stimulation is associated with apoptosis regression in the amygdala following myocardial infarction. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC).     

Tumor necrosis factor-alpha participates in apoptosis in the limbic system after myocardial infarction

This study was designed to determine the role of tumor necrosis factor-alpha (TNFα) in apoptosis observed in the myocardium and limbic system after myocardial ischemia. PEG sTNFRI, a recombinant, human, soluble p55 Type 1 TNF receptor (3 mg/kg) or vehicle (saline) was administered s.c. to male Sprague-Dawley rats on days 5, 3 and 1 before myocardial ischemia. The animals were then subjected, under anesthesia, to left anterior descending coronary artery occlusion for 40 min, followed by 15-min or 72-h reperfusion. Caspase-3 and -8 activities as well as terminal dUTP nick-end labelling-positive cells were examined in the myocardium (subendocardial and subepicardial regions), lateral (LA) and medial amygdala (MA) and hippocampus (CA1, CA3, dentate gyrus (DG)). After 15 min of reperfusion, the subendocardial and CA1 regions presented an increase in caspase-3 activity, whereas caspase-8 activity appeared to be augmented in the DG. PEG sTNFRI inhibited caspase-8 activation in the DG. After 72 h of reperfusion, plasma TNFα levels were reduced in the treated groups. The DG, CA1, CA3 and MA showed an increment of caspase-8 activity, which was reversed by PEG sTNFRI, except in the MA. Furthermore, caspase-3 activity was increased in the CA1, DG, LA and MA. These results indicate that TNFα contributes to apoptosis via activation of the extrinsic pathway in the limbic system after myocardial infarction, which is not the case in the myocardium.     

Effects of different dietary ω-6/3 polyunsaturated fatty acids ratios on infarct size and the limbic system after myocardial infarction

Changes in dietary omega-6/3 polyunsaturated fatty acids (PUFA) ratios affect anti- and proinflammatory equilibrium. As reperfused myocardial infarction (MI) is an inflammatory pathology that alters the cell integrity of the myocardium but also of other tissues, such as the hippocampus and amygdala, attenuation of the inflammation could be helpful in maintaining cell integrity after MI. Therefore, we hypothesized that a decrease in the dietary omega-6/3 PUFA ratio, without altering the diet content in total fat, proteins, or carbohydrates, will result in a reduction of infarct size and a diminution of postreperfusion apoptosis observed in the amygdala and hippocampus. Male Sprague-Dawley rats were fed 1 of 3 diets containing different omega-6/3 PUFA ratios for 2 weeks (5:1; 1:1; 1:5). Then, myocardial ischemia was induced by left anterior descending coronary artery occlusion for 40 min, followed by reperfusion. Cardioprotective mechanisms were studied in the myocardium at 15 min of reperfusion, along with myocardial infarct size after 24 h of reperfusion. Apoptosis was evaluated in the hippocampus and the amygdala. We found that infarct size was significantly reduced by 32% in groups 1:5 and 1:1 vs. group 5:1. Akt activity was higher in groups 1:5 and 1:1 compared with group 5:1. Caspase-3 enzymatic activity doubled in area CA1 and the dentate gyrus (DG) in group 5:1 compared with groups 1:1 and 1:5. In addition, caspase-8 enzymatic activity was increased in the DG at 24 h, and caspase-9 was enhanced in CA1 at 24 h in group 5:1 vs. groups 1:1 and 1:5. These results demonstrate that the increase in the dietary omega-3 PUFA, at the expense of omega-6 PUFA, reduces infarct size and helps to inhibit apoptosis in the limbic system after MI.     

5-HT2 receptor blocker sarpogrelate prevents downregulation of antiapoptotic protein Bcl-2 and protects the heart against ischemia-reperfusion injury

Even though reperfusion is the treatment of choice in patients admitted with acute myocardial infarction, reperfusion itself has been demonstrated to activate various pathological factors especially following procedures of cardiac revascularization. 5-hydroxytryptamine (5HT) is one such factor activated during reperfusion and is known to trigger the post ischemic contractile dysfunction and pathological apoptosis. Here we demonstrate the potential effects of the 5-HT(2)A antagonist sarpogrelate in protecting the myocardium against reperfusion injury of heart. Male Wistar rats weighing between 220 and 240 g were subjected to 30 min left coronary artery (LCA) occlusion and 120 min reperfusion. Sarpogrelate (4 mg/kg) was infused intravenously for 30 min either before LCA occlusion or at reperfusion. Following reperfusion the samples were collected for infarction area, immunohistochemistry, western blotting and myocardial metabolite analysis. Sarpogrelate infusion before ischemia resulted in (a) significant recovery of post ischemic cardiac functions (LVDP, EDP), (b) significant reduction in the infarct size among the risk area after triphenyl tetrazolium chloride staining (p<0.001), (c) decreased tissue water content (p<0.05), (d) well preserved myocardial ATP (p<0.05), (e) reduction in Bcl-2 downregulation and caspase 3 activation and (g) less prevalence of apoptotic cells (3.1+/-0.4% to 15.2+/-0.6%, drug versus control). Treating the rats with sarpogrelate during reperfusion also showed similar results. This study thus demonstrates the protective effects of sarpogrelate and supports the role for 5-HT2A inhibition in preventing the reperfusion injury of the heart.     

LOX-1 pathway affects the extent of myocardial ischemia-reperfusion injury

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was originally identified as a receptor for oxidized low-density lipoprotein predominantly expressed in endothelial cells. LOX-1 expression can be induced in cardiomyocytes and that activation of LOX-1 is involved in apoptosis. To investigate possible roles of LOX-1 in myocardial ischemia-reperfusion injury, rats were subjected to coronary artery ligation for 1h followed by reperfusion for 2h. Immunohistochemistry revealed that expression of LOX-1 in cardiac myocytes was induced following ischemia-reperfusion but not ischemia alone. Administration of anti-LOX-1 monoclonal antibody resulted in a nearly 50% reduction in myocardial infarction size compared with that of normal IgG or saline (P<0.05). These findings suggest that activation of the LOX-1 pathway is involved in determining the extent of myocardial ischemia-reperfusion injury and that inhibition of the LOX-1 pathway may provide a novel strategy for treatment of acute myocardial infarction in humans.     

Early anti-apoptosis treatment reduces myocardial infarct size after a prolonged reperfusion

OBJECTIVE : Significant myocardial apoptosis occurs in ischemia/reperfused hearts. However, the contribution of apoptosis to the development of myocardial injury remains controversial. The present study attempted to obtain evidence that inhibition of apoptosis at early reperfusion can reduce myocardial infarction after prolonged reperfusion. METHODS : Adult male rats were subjected to 30 min ischemia and 4 (apoptosis assay) or 24 h (myocardial infarction determination) of reperfusion and treated with vehicle, SB 239063, insulin or insulin plus wortmannin. RESULTS : Treatment with SB 239063 or insulin markedly decreased myocardial apoptosis (10.6 +/- 1.5% and 7.9 +/- 0.9% respectively, P < 0.01 vs. vehicle) and significantly reduced infarct size (43 +/- 3.6% and 35 +/- 2.9%, respectively, P < 0.01 vs. vehicle). Most interestingly, inhibition of insulin signaling with wortmannin to block insulin signaling not only blocked insulin's anti-apoptotic effect, but also abolished its infarct reduction property. CONCLUSION : These data indicate that apoptosis contributes to the development of myocardial infarction, and inhibition of apoptosis at early reperfusion reduces the myocardial infarction.     

Emodin-mediated protection from acute myocardial infarction via inhibition of inflammation and apoptosis in local ischemic myocardium

Acute myocardial infarction (AMI) is associated with inflammation and apoptosis. Emodin plays an anti-inflammatory role in several inflammatory diseases. Recent studies have demonstrated that emodin protects against myocardial ischemia/reperfusion injury. However, its mechanism underlying its effects remains unknown. In a murine model of AMI, based on ligation of the left coronary artery, administration of emodin reduced myocardial infarct size (MIS) in a dose-dependent manner. Emodin significantly suppressed TNF-alpha expression and NF-kappaB activation in the local myocardial infarction area. Treatment with emodin inhibited myocardial cell apoptosis by inhibiting caspase-3 activation. Therefore, these studies demonstrate that emodin protects against myocardial cell injury via suppression of local inflammation and apoptosis.     

Reduction of myocardial infarct size by fluvastatin

Statins have a variety of cardioprotective properties following chronic treatment. In contrast, little is known about the acute effects. Reperfusion acutely injures the heart by activation of neutrophils as well as endothelial cells. Because statins are known to influence the processes pathogenetically involved, we hypothesized that acute application of statins attenuates the sequelae of cardiac reperfusion. In rats, myocardial infarction (MI) was induced by ligature of the left coronary artery followed by reperfusion. Myocardial blood flow (MBF) was determined by H2 clearance and regional myocardial function (fractional thickening, FT) by pulsed Doppler. MI size was measured by triphenyltetrazolium chloride (TTC) staining, neutrophil extravasation by determination of myeloperoxidase (MPO) activity, and nitric oxide generation via measurement of cGMP. Treatment with fluvastatin, administered intravenously 20 min before the onset of ischemia, significantly attenuated the decline of FT and MBF at the end of the reperfusion period and significantly reduced MI size. Furthermore, fluvastatin induced a significant reduction of MPO activity and an increase of cGMP level compared with the control group. The effect of fluvastatin was completely abolished following pretreatment of NG-nitro-l-arginine methyl ester (l-NAME). These findings suggest that acute application of fluvastatin reduces MI size and attenuates reperfusion injury. We propose that the underlying mechanism is at least partially an inhibition of inflammation and endothelial dysfunction by preventing the activation and extravasation of neutrophils.     

Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways

12/15-Lipoxygenase (LOX) is a member of the LOX family that catalyzes the step from arachidonic acid to hydroxy-eicosatetraenoic acids (HETEs). Previous studies demonstrated that 12/15-LOX plays a critical role in the development of atherosclerosis, hypertension, heart failure, and other diseases; however, its role in myocardial ischemic injury was contraversal. Here, we investigated the inhibition of 12/15-LOX by baicalein on acute cardiac injury and dissected its molecular mechanism. In a mouse model of acute ischemia/reperfusion (I/R) injury, 12/15-LOX was significantly upregulated in the peri-infarct area surrounding the primary infarction. In cultured cardiac myocytes, baicalein suppressed apoptosis and caspase 3 activity in response to simulated ischemia/reperfusion (I/R). Moreover, administration of 12/15-LOX inhibitor, baicalein, significantly attenuated myocardial infarct size induced by I/R injury. Moreover, baicalein treatment significantly inhibited cardiomyocyte apoptosis, inflammatory responses and oxidative stress in the heart after I/R injury. The mechanisms underlying these effects were associated with the activation of ERK1/2 and AKT pathways and inhibition of activation of p38 MAPK, JNK1/2, and NF-kB/p65 pathways in the I/R-treated hearts and neonatal cardiomyoctes. Our data indicated that 12/15-LOX inhibitor baicalein can prevent myocardial I/R injury by modulation of multiple mechanisms, and suggest that baicalein could represent a novel therapeutic drug for acute myocardial infarction.     

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.     

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.     

Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium

Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 +/- 3.8%; 5 microM fasudil, 18.2 +/- 3.1%; 0.3 microM Y-27632, 19.4 +/- 4.4%; 5 microM Y-27632, 9.2 +/- 2.9%). When 5 microM Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 +/- 2.6% vs. control 33.4 +/- 4.4%, P<0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 +/- 6.1%) and N(omega)-nitro-L-arginine methyl ester (30.4 +/- 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, suggesting that ROCK activation at reperfusion may be deleterious through suppression of the RISK pathway.     

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

瞬时受体电位香草酸亚型1激活通过抑制线粒体通透性转换孔开放保护急性心肌梗死小鼠的心肌细胞抗凋亡

瞬时受体电位香草酸亚型1（TRPV1）在心肌缺血激活后可传导心绞痛信号,释放神经肽,减轻心肌梗死后的心肌细胞凋亡。目前,TRPV1激活抑制心肌梗死后细胞凋亡的具体机制尚不清楚。线粒体通透性转换孔（MPTP）的开放与心肌细胞缺血再灌注损伤密切相关,抑制其开放可保护心肌缺血后的心肌细胞抗凋亡。本研究证明,TRPV1激活通过抑制MPTP开放而减少心肌细胞凋亡。首先,本研究利用左冠状动脉前降支结扎术建立了TRPV1基因敲除（TRPV1-/-）和野生型（WT）小鼠心肌梗死模型,辅以环孢素A（CSA）预处理抑制 MPTP开放,比较观察TRPV1、MPTP在心肌梗死中的作用。心肌组织切片氯化三苯基四氮唑（TTC）染色显示,心肌缺血24 h,TRPV1-/-小鼠的心肌梗死面积明显大于WT型小鼠。而经CSA预处理的TRPV1-/-小鼠比TRPV1-/-小鼠梗死面积明显减小。TUNEL检测心肌细胞凋亡指数（AI）揭示,WT型心肌梗死小鼠的AI明显低于TRPV1-/- 心肌梗死小鼠,而CSA预处理明显降低TRPV1-/-小鼠心肌细胞的AI。Western印迹检测胱天蛋白酶3、胱天蛋白酶9、Bcl-2、Bax、p53和细胞色素C（Cyt-C）水平。结果证明,TRPV1的激活可抑制MPTP的开放,减少线粒体Cyt-C的外溢,降低胱天蛋白酶9和胱天蛋白酶3的表达。GENMEN光度法检测MPTP开放实验显示,激活的TRPV1明显抑制了MPTP的开放。本研究证实,急性心肌梗死后的TRPV1激活可能通过抑制MPTP开放而抵抗心肌细胞凋亡,对心肌起保护作用。     

Transduction of anti-cell death protein FNK protects isolated rat hearts from myocardial infarction induced by ischemia/reperfusion

Artificial anti-cell death protein FNK, a Bcl-x(L) derivative with three amino acid-substitutions (Y22F, Q26N, and R165K) has enhanced anti-apoptotic and anti-necrotic activity and facilitates cell survival in many species and cell types. The objectives of this study were (i) to investigate whether the protein conjugated with a protein transduction domain (PTD-FNK) reduces myocardial infarct size and improves post-ischemic cardiac function in ischemic/reperfused rat hearts, and (ii) to understand the mechanism(s) by which PTD-FNK exerts a protective effect. Isolated rat hearts were subjected to 35-min global ischemia, followed by 120-min reperfusion using the Langendorff methods. PTD-FNK (a total of 30 microl) was injected intramuscularly into the anterior wall of the left ventricle either at 1 min after induction of global ischemia (group A) or at 30 min after induction of global ischemia (at 5 min before reperfusion) (group B). In group A, infarct size was significantly reduced from 47.8+/-6.8% in the control to 30.4+/-5.2, 28.7+/-3.8, and 30.4+/-6.8% with PTD-FNK at 5, 50, and 500 nmol/l, respectively (p<0.05). Temporal recovery of left ventricular developed pressure at 60 min and 120 min after reperfusion was significantly better in PTD-FNK (50 and 500 nmol/l)-treated groups than in the control (p<0.05). In contrast, PTD-FNK treatment had no effect on group B. Western blot analysis showed that PTD-FNK markedly inhibited procaspase-3 cleavage (activation of caspase-3) and reduced the number of nuclei stained by a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphoshate nick-end labeling (TUNEL) assay. These findings suggest that PTD-FNK reduces the volume of myocardial infarction with corresponding functional recovery, at least in part, through the suppression of myocardial apoptosis following ischemia/reperfusion.     

下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。     

Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury

To learn whether nitric oxide (NO) inhalation can decrease myocardial ischemia-reperfusion (I/R) injury, we studied a murine model of myocardial infarction (MI). Anesthetized mice underwent left anterior descending coronary artery ligation for 30, 60, or 120 min followed by reperfusion. Mice breathed NO beginning 20 min before reperfusion and continuing thereafter for 24 h. MI size and area at risk were measured, and left ventricular (LV) function was evaluated using echocardiography and invasive hemodynamic measurements. Inhalation of 40 or 80 ppm, but not 20 ppm, NO decreased the ratio of MI size to area at risk. NO inhalation improved LV systolic function, as assessed by echocardiography 24 h after reperfusion, and systolic and diastolic function, as evaluated by hemodynamic measurements 72 h after reperfusion. Myocardial neutrophil infiltration was reduced in mice breathing NO, and neutrophil depletion prevented inhaled NO from reducing myocardial I/R injury. NO inhalation increased arterial nitrite levels but did not change myocardial cGMP levels. Breathing 40 or 80 ppm NO markedly and significantly decreased MI size and improved LV function after ischemia and reperfusion in mice. NO inhalation may represent a novel method to salvage myocardium at risk of I/R injury.     

Remote ischaemic preconditioning and prevention of cerebral injury

Bilateral carotid artery occlusion of 10 min followed by reperfusion for 24 hr was employed in present study to produce ischaemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Short-term memory was evaluated using elevated plus maze. Inclined beam walking test was employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired short-term memory, motor co-ordination and lateral push response. A preceding episode of mesenteric artery occlusion for 15 min and reperfusion of 15 min (remote mesenteric ischaemic preconditioning) prevented markedly ischaemia-reperfusion-induced cerebral injury measured in terms of infarct size, loss of short-term memory, motor coordination and lateral push response. Glibenclamide (5 mg/kg, iv) a KATP channel blocker and caffeine (7 mg/kg, iv) an adenosine receptor blocker attenuated the neuroprotective effect of remote mesenteric ischaemic preconditioning. It may be concluded that neuroprotective effect of remote mesenteric ischaemic preconditioning may be due to activation of adenosine receptors and consequent activation of KATP channels in mice.     

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3^−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3^−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3^−/− mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.