Hyperbaric oxygen preconditioning alleviates myocardial ischemic injury in rats

It has been shown that after ischemia-reperfusion, application of hyperbaric oxygen (HBO) reduces cardiac injury. In this study we tested the hypothesis that HBO preconditioning reduces injury to the ischemic myocardium. One hundred and eight adult male Sprague-Dawley rats (250-280 g) were randomly divided into four groups: normoxia + sham surgery (CS), normoxia + permanent occlusion of the left anterior descending (LAD) coronary artery (CMI), HBO preconditioning + sham surgery (HS), and HBO preconditioning + permanent LAD occlusion (HMI). Rats receiving HBO preconditioning were intermittently exposed to 100% O(2) at 2.5 atmosphere absolute (ATA) for 60 min, twice daily for 2 days followed by 12 hrs of recovery in room air prior to the myocardial ischemic insult induced by LAD ligation. Rats in the normoxia group were time-matched with the HBO group and maintained under normoxic conditions prior to LAD occlusion. At 3 and 7 days after LAD occlusion, heart function parameters were measured by inserting a catheter into the left ventricle, infarct size was calculated using the method of TTC staining, myocardial capillary density was determined by immunohistochemical staining with a monoclonal anti-CD(31)/PECAM-1 antibody, and VEGF protein level was determined by Western blot analysis. At 3 days after LAD ligation, the infarct size of the HMI group was significantly smaller than that of the CMI group (26 +/- 2.5% vs. 38 +/- 3%, P < 0.05). The heart function parameters including left ventricular systolic pressure (LVSP), +dP/dt(max) and -dP/dt(max) were significantly improved in the HMI group compared to the CMI group at 3 and 7 days after LAD occlusion. Capillary density and VEGF protein levels were significantly increased in the ischemic myocardium pre-exposed to HBO. We conclude that HBO preconditioning alleviates myocardial ischemia in rat model.     

Preconditioning of heart by repeated stunning. Adaptive modification of antioxidative defense system.

Previous studies demonstrated that preconditioning of a heart by repeated stunning can reduce the cellular injury to the heart from subsequent acute ischemic insult. To examine the possible biochemical mechanism for such myocardial preservation afforded by preconditioning, swine heart was subjected to four episodes of 5 min. stunning by occluding the left anterior descending coronary artery (LAD), followed by 10 min. of reperfusion after each stunning. Heart was then made regionally ischemic for 60 min. by LAD occlusion, followed by 6 hrs. reperfusion. Control heart was perfused for 60 min., followed by 60 min. ischemia and 6 hrs. reperfusion. The results of our studies indicated the stimulation of a number of antioxidative enzymes, including Mn-superoxide dismutase (Mn-SOD), catalase, glutathione peroxidase, and glutathione reductase, after repeated stunning and reperfusion. In addition, a number of new proteins were expressed after preconditioning the heart, including some oxidative-stress related proteins and 72 kDa heat-shock protein. These results suggest that preconditioning of a heart by repeated stunning may lead to strengthening of the oxidative defense system of the heart, which is likely to play a role in myocardial preservation during subsequent ischemic and reperfusion injury.     

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available ^1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important ^{7, 14, 30, 39}. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP ³⁹. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds³⁹. In addition to infarct staining, we tested cardiac troponin I (cTnI) as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction^{6, 8, 30, 40, 41}. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.     

Effect of desflurane-induced preconditioning following ischemia-reperfusion on nitric oxide release in rabbits

Nitric oxide (NO) is the mediator of ischemic preconditioning against myocardial infarction. Desflurane produces anesthetic preconditioning to protect the myocardium against infarction. In the model of myocardial ischemia-reperfusion injury in rabbits, we evaluated desflurane-induced ischemic preconditioning and studied its mechanism of NO synthesis. Thirty-two male adult New Zealand white rabbits were anesthetized with intravenous (IV) 30 mg/kg pentobarbital followed by 5 mg/kg/hr infusion. All rabbits were subjected to 30 minutes (min) long lasting left anterior descending coronary artery (LAD) occlusion and three hours (hr) of subsequent reperfusion. Before LAD occlusion, the rabbits were randomly allocated into four groups for preconditioning treatment (eight for each group). The control group did not receive any preconditioning treatment. The desflurane group received inhaled desflurane 1.0 MAC (minimal end-tidal alveolar concentration) for 30 min that was followed by a 15 min washout period. The L-NAME-desflurane group received L-NAME (NG-nitro-L-arginine methyl ester; non-selective Nitric Oxide Synthetase (NOS) inhibitor) 1 mg/kg IV 15 min before 1.0 MAC inhaled desflurane for 30 min. The L-NAME group received L-NAME 1 mg/kg IV. Infarct volume, ventricular arrhythmia, plasma lactate dehydrogenase (LDH), creatine kinase (CK) activity and myocardial perfusion were recorded simultaneously. We have found that hemodynamic values of the coronary blood flow before, during, and after LAD occlusion were not significantly different among these four groups. For the myocardial ischemia-reperfusion injury animals, the infarction size (mean +/- SEM) in the desflurane group was significantly reduced to 18 +/- 3% in the area at risk as compared with 42 +/- 7% in the control group, 35 +/- 6 in the L-NAME group, and 34 +/- 4% in the L-NAME-desflurane group. The plasma LDH, CK levels, and duration of ventricular arrhythmia were also significantly decreased in the desflurane group during ischemia-reperfusion injury. Our results indicate that desflurane is an anesthetic preconditioning agent, which could protect the myocardium against the ischemia-reperfusion injury. This beneficial effect of desflurane on the ischemic preconditioning is probably through NO release since L-NAME abrogates the desflurane preconditioning effect.     

Ischemic preconditioning protects cardiomyocytes against ischemic injury by inducing GRP78

Ischemic preconditioning (IP) conferred by brief ischemia-reperfusion induces resistance to cell injury due to the following lethal ischemia. This study aimed to elucidate whether 78-kDa glucose-regulated protein (GRP78), a main ER molecular chaperone, contributes to IP-mediated protection against ischemic myocardial injury. In a rat coronary artery occlusion model, the GRP78 protein level increased to 210% of the sham level by early IP with three cycles of 4-min ischemia and 4-min reperfusion. The IP reduced infarct size in subsequent lethal ischemia. In primary cardiomyocytes, the simulated IP procedure, incubation in oxygen-glucose deprivation (OGD) medium, also increased the GRP78 expression and suppressed the cell death caused by lethal ischemia. Transfection of grp78 antisense oligonucleotide attenuated the IP-mediated resistance to ischemia. This study showed for the first time that early IP up-regulates myocardial GRP78. It was suggested that GRP78 induced by early IP contributes to protect cardiomyocytes against ischemic injury.     

Coronary reactive hyperaemia after two periods of coronary occlusion in the anaesthetized goat.

In the coronary circulation an ischaemic preconditioning obtained with two periods of 2.5 min each of occlusion of the left circumflex coronary artery alters the pattern of a coronary reactive hyperaemia which follows 15 s only of occlusion of the studied artery. The most remarkable change consists of a reduction of 40-45% of the time required by the flow to reach the maximum hyperaemic peak (time to peak) after the brief occlusion. The present investigation was planned to study whether the time to peak of the hyperaemia following the second 2.5 min preconditioning occlusion was shorter than the hyperaemia following the first occlusion. Experiments performed in the anaesthetized goat, in which coronary flow was measured with an electromagnetic flow-probe placed around the left circumflex coronary artery showed that in the hyperaemia occurring after the second preconditioning occlusion the time to peak was reduced by 18% only. The moderate effect of the second preconditioning occlusion in reducing the time to peak is attributed to the fact that the heart was already partially preconditioned after the first occlusion and that after relatively long periods (2.5 min) of occlusion the metabolic component of the hyperaemic response was so predominant to partially mask the role of the vascular mechanisms presumably responsible for the reduction of the time to peak.     

The effects of ischemic preconditioning on resting coronary flow and reactive hyperemia: involvement of A1 adenosine receptors

During the myocardial protection induced by ischemic preconditioning a reduction in myocardial metabolism occurs due to activation of the A1 adenosine receptors. This study investigates whether preconditioning changes both resting coronary flow and the magnitude of coronary reactive hyperemia and whether A1 adenosine receptors are involved in the observed changes. Experiments were performed in 14 goats (30-50 kg body weight). After the animals were anesthetized with ketamine, an electromagnetic flow-probe was used to record blood flow in the left circumflex coronary artery. Distal to the probe, an occluder was placed to produce ischemic preconditioning and reactive hyperemia. Preconditioning was obtained with two periods of 2.5 min of coronary occlusion separated from each other by 5 min of reperfusion. Coronary reactive hyperemia was obtained with 15 s of occlusion of the artery before and after preconditioning. In a group of goats before preconditioning 0.2 mg kg(-1) of 8-cyclopentyl-dipropylxanthine (CPX), an A1 adenosine receptor blocker, were given intravenously. In all animals ischemic preconditioning did not alter resting coronary flow, but, in the absence of A1 adenosine receptor blockade, reduced the reactive hyperemic response. The total hyperemic flow and the excess/debt flow ratio were reduced by about 25% and 30% respectively. The A1 adenosine receptor blockade "per se" did not cause any change in the resting flow and in the parameters of the reactive hyperemia. Unlike what observed in the absence of blockade, after CPX ischemic preconditioning was unable to reduce total hyperemic flow and the excess/debt flow ratio. The results suggest that ischemic preconditioning reduces the coronary hyperemic response by decreasing the myocardial metabolism through the activation of the A1 adenosine receptors.     

Assessment of collateral artery function and growth in a pig model of stepwise coronary occlusion

Therapeutic stimulation of collateral artery growth is a promising approach for treatment of cardiovascular diseases. Unfortunately, translation into clinical practice yet remains cumbersome. Cardiovascular physiology and anatomy are major determinants of vascular growth processes. Hence, large-animal models are needed to improve clinical translatability of preclinical research. Furthermore, acute complete occlusions are mostly applied in experimental research, whereas stepwise occlusions are more often observed in human disease. We developed a model of coronary collateral artery growth in which 1) the artery is occluded in a step wise approach, and 2) effects of local treatment can be measured individually for each supplying coronary vessel. A hemodynamically relevant stenosis was created by implantation of a tapered stent at day 0 (d0) in the left circumflex artery (LCX), followed by complete arterial occlusion at day 14 (d14). Fluorescent microspheres were injected for demarcation of perfusion territories at each time point. Three and four weeks after induction of stenosis, collateral conductance measurements were performed for each coronary artery separately using differently labeled fluorescent microspheres. Postmortem angiography after acute LCX occlusion confirmed the presence of preexistent coronary anastomoses in the pig. The tapered stent created a hemodynamically significant stenosis immediately postplacement (fractional flow reserve, 0.70 ± 0.03). Between day 21 and 28, collateral conductance significantly increased in both the left anterior descending (LAD) and the right coronary artery (RCA)-supplied, collateral-dependent territories (LAD d21, 0.77 ± 0.14; LAD d28, 1.35 ± 0.12; RCA d21, 0.88 ± 0.29; RCA d28, 1.70 ± 0.16 ml · min(-1) · g(-1) · 100 mmHg(-1)), indicating collateral artery growth. We here describe a new translational minimally invasive model of coronary collateral artery growth in pigs, according to a defined protocol of LCX-stenosis and subsequent occlusion, allowing preclinical evaluation of arteriogenic therapies.     

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning

The role of prostaglandins in the antiarrhythmic effect of ischemic preconditioning (IP) was investigated in pentobarbital-anesthetized rats. In 5 unpreconditioned control rats, 30 min of occlusion of the left coronary artery elicited ventricular tachycardia (VT) and fibrillation (VF), with an average duration of VT and VF of 51 +/- 6 and 43 +/- 4 s, respectively. Frequent ventricular premature beats (VPBs; average 1,249 +/- 145) were also documented in these animals. Thirty minutes of reperfusion after the prolonged coronary occlusion in these animals caused more severe arrhythmias, including irreversible VF. In animals pretreated with IP (n = 5), which was achieved by 3 cycles of 3 min of occlusion followed by 5 min of reperfusion, 30 min of coronary artery occlusion caused neither VT nor VF, but occasional VPBs (average 2 +/- 1, p < 0.001 vs. control). Only occasional VPBs were observed during 30 min of reperfusion in this group. In animals pretreated with indomethacin (1 mg/kg i.v., n = 5) followed by IP, prolonged ischemia and reperfusion led to frequent VPBs but no VT or VF. The average number of VPBs during ischemia and reperfusion in this indomethacin-treated group was less than that of the controls but greater than the IP-only group (p < 0.01). In conclusion, prostaglandins appear to play a role in the protective effect of IP against VPBs during acute ischemia and reperfusion.     

The role of adenylate cyclase in ischemic preconditioning in the rat heart: a cytochemical study

Using catalytic cytochemistry the AC activity was studied during ischemic preconditioning (IP) (5 min occlusion of LAD and 10 min reperfusion) followed by 30 min regional ischemia in isolated Langendorff-perfused rat heart. In controls the specific precipitate of AC reaction was found on the sarcolemma (SL) and the junctional sarcoplasmic reticulum (JSR) of cardiomyocytes. After prolonged ischemia the reaction product was absent, whereas IP followed by prolonged ischemia protected the AC activity on SL and JSR. IP-induced enhancement of AC activity in this model was accompanied by significant reduction of ischemia/reperfusion fibrillation. The results suggest involvement of AC system in mechanisms of IP.     

大鼠心肌缺血预适应诱导表达上调基因的筛选和鉴定

采用反复短时间结扎及松解左冠状动脉前降支构建大鼠心肌缺血预适应动物模型,运用抑制消减杂交技术建立大鼠心肌缺血预适应诱导表达上调基因的消减cDNA文库,通过反向RNA点杂交对部分文库基因进行差异表达初筛,选取表达差异最明显的85个基因进行测序,获得了31个核编码基因和18个新基因（EST）,核编码基因中有相当一部分与细胞保护或信号转导有关,新基因已被GenBank收录.从已测序基因中任选5个进行RT-PCR检测,并任选2个进行RNA印迹检测,均证实在缺血预适应时表达增高.上述结果为进一步深入研究缺血预适应心肌保护作用的分子机制和克隆新的缺血预适应相关基因提供了重要信息.     

Development of a preclinical model of ischemic cardiomyopathy in swine

A number of promising therapies for ischemic cardiomyopathy are emerging, and the role of translational research in testing the efficacy and safety of these agents in relevant clinical models has become important. The goal of this study was to develop a chronic model of ischemic cardiomyopathy in a large animal model. In this study, 40 consecutive pigs were initially enrolled. To induce progressive stenosis, a plastic occluder with a fixed diameter of 1.0 mm fitted with an 18-gauge copper wire was placed around the proximal left anterior descending (LAD) coronary artery. Coronary angiography, hemodynamic measurements, and echocardiography were performed at 2 wk and 1, 2, and 3 mo. Overall mortality was 26% at 3 mo, and up to 80% of the pigs showed total occlusion of LAD at 1 mo. A significant depression of peak LV pressure rate of rise (+dP/dt(max)) was observed in the animals showing total artery occlusion throughout the study. Left ventricular ejection fraction was also impaired, and the left ventricular volumes tended to be larger in the pigs with occlusion. Approximately 10% of scar tissue was found in the LAD occluded pigs, whereas the coronary flow pattern in the rest of the area took the pattern of hibernating myocardium. At the same time, histological and protein analysis established the presence of fibrosis and ongoing apoptosis in the ischemic area. In this model, the timing and incidence of total occlusion and low mortality offer significant advantages over other ischemic cardiomyopathy models in conducting preclinical studies.     

No ischemic preconditioning in heterozygous connexin43-deficient mice

Protein kinase Cepsilon (PKCepsilon) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCepsilon colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 +/- 3.4% vs. 23.7 +/- 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 +/- 6.0% vs. 25.1 +/- 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 +/- 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.     

Non-ischemic myocardial preconditioning

The reduction of infarct size produced by brief ischemic episodes prior to a sustained occlusion of a coronary artery, called ischemic preconditioning, is a well known phenomenon that occurs in several species, but its mechanism is still under investigation. Recent reports support the idea that this protection can also be obtained by non-ischemic maneuvers like distention of the left ventricle and metabolic stimulation of myocardial cells. The features of non-ischemic preconditioning (temporal limitation, second window, tolerance development, remote preconditioning and efficiency of the protection), as opposed to those of ischemic preconditioning, are still to be determined. Neither is it known if non-ischemic preconditioning occurs in humans. From a physiological point of view the protective effect of an increase in metabolic rate of the heart means a constant feed-back mechanism in the myocardial cell that counteracts the presumptive damage consequent to the increase in metabolism. Therefore, in the presence of a sudden coronary occlusion the metabolic rate of the heart immediately before the occlusion would have a dual role of increasing the degree of ischemia and of protecting against it.     

Effect of age on alpha-calcitonin gene-related peptide-mediated delayed cardioprotection induced by intestinal preconditioning in rats

In the present study, we examined whether age-related reduction in cardioprotection of intestinal ischemic preconditioning is related to stimulation of the release and synthesis of calcitonin gene-related peptide (CGRP) in rats. Ischemia-reperfusion injury was induced by a 45-min coronary artery occlusion and 180-min reperfusion, and ischemic preconditioning was induced by six cycles of 4-min ischemia and 4-min reperfusion of the small intestine. The serum concentration of creatine kinase, infarct size, the expression of CGRP isoforms (alpha- and beta-CGRP) mRNA in lumbar dorsal root ganglia and CGRP concentration in plasma were measured. Pretreatment with intestinal ischemic preconditioning for 24 h significantly reduced infarct size and creatine kinase release concomitantly with a significant increase in the expression of alpha-CGRP mRNA, but not beta-CGRP mRNA, and plasma concentrations of CGRP at 6 months of age but not at 24 months of age. These results suggest that the delayed cardioprotective effect of intestinal ischemic preconditioning is decreased in senescent rats, and the age-related change is related to reduction of the synthesis and release of alpha-CGRP.     

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10 min followed by different amounts of reperfusion time (1-24 h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24 h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE₂ increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.