The protective effects and potential mechanism of Calpain inhibitor Calpeptin against focal cerebral ischemia–reperfusion injury in rats

To demonstrate the protective effects of Calpeptin as the Calpain inhibitor against focal cerebral ischemia–reperfusion injury in rats and to explore it’s possible mechanism. 96 rats were randomly divided into four groups. The model of middle cerebral artery occlusion was used for the research of focal cerebral ischemia. Using this animal model, the effects of Calpeptin on the neurological functions, infarction volume and infarction volume percentage of brain, Caspase-3 expression and neuronal apoptosis in hippocampal CA1 sector after focal cerebral ischemia–reperfusion injury in rats were investigated. The current results confirmed that Calpeptin as the Calpain inhibitor might paly an important role for neuroprotection against focal cerebral ischemia–reperfusion injury. Calpeptin could reduce the neuronal apoptosis in hippocampal CA1 sector when the rats was subjected to the focal cerebral ischemia–reperfusion, the potential mechanism might be related to the inhibition of the expression of Caspase-3 by Calpeptin. However, it is still unknown to what the exact mechanism of Calpeptin inhibits the activation of Caspase-3 in this process. Therefore, further research needs to be done to unravel the underlying mechanisms in the future.     

Sphingolipid therapy in myocardial ischemia–reperfusion injury

Sphingolipids are known to play a significant physiological role in cell growth, cell differentiation, and critical signal transduction pathways. Recent studies have demonstrated a significant role of sphingolipids and their metabolites in the pathogenesis of myocardial ischemia–reperfusion injury. Our laboratory has investigated the cytoprotective effects of N,N,N-trimethylsphingosine chloride (TMS), a stable N-methylated synthetic sphingolipid analogue on myocardial and hepatic ischemia–reperfusion injury in clinically relevant in vivo murine models of ischemia–reperfusion injury. TMS administered intravenously at the onset of ischemia reduced myocardial infarct size in the wild-type and obese (ob/ob) mice. Following myocardial I/R, there was an improvement in cardiac function in the wild-type mice. Additionally, TMS also decreased serum liver enzymes following hepatic I/R in wild-type mice. The cytoprotective effects did not extend to the ob/ob mice following hepatic I/R or to the db/db mice following both myocardial and hepatic I/R. Our data suggest that although TMS is cytoprotective following I/R in normal animals, the cytoprotective actions of TMS are largely attenuated in obese and diabetic animals which may be due to altered signaling mechanisms in these animal models. Here we review the therapeutic role of TMS and other sphingolipids in the pathogenesis of myocardial ischemia–reperfusion injury and their possible mechanisms of cardioprotection.     

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.     

GnRH analogue attenuated apoptosis of rat hippocampal neuron after ischemia–reperfusion injury

The expression and new functions of reproductive hormones in organs beyond hypothalamus-pituitary-gonad axis have been reported. So far, there is no report about the protective effects of GnRH analogue to hippocampal neurons suffering from ischemia–reperfusion injury. Middle cerebral artery occlusion model together with TUNEL staining were made in vivo and oxygen-glucose deprivation model together with double staining of Annexin V/PI with flow cytometer were made in vitro to observe the anti-apoptotic effects of GnRH analogue to hippocampal neurons after ischemia–reperfusion injury. The results found that the number of TUNEL positive pyramidal neurons in CA1 region in GnRH analogue experiment group was less than that in control group in vivo; the percentage of apoptotic neurons in GnRH analogue experiment group was less than that in control group in vitro. These findings suggested that pretreatment with certain concentration of GnRH analogue could attenuate apoptosis of hippocampal neurons. GnRH analogue has the protective effects to neurons.     

小型猪心肌梗死介入治疗后无复流动物模型的制备

目的制备猪急性心肌梗死冠状动脉介入治疗（AMI—PCI）后无复流（no—reflow）动物模型。方法五指山小型猪26头,行左、右冠状动脉造影和左心室造影,记录有创血流动力学参数,通过球囊闭塞、微血栓注入造成左前降支无复流,监测体表和冠脉内心电图变化。结果制模共有21头猪成活,19头达到AMI—PCI后无复流动物模型标准,即TIMI血流≤2级,校正的TIMI血流记帧法（CTFC）≥36．2帧,制模成功率73．1％。无复流模型建立成功后心率增快,血压下降,心肌耗氧量增加,左心室舒张期末压和肺毛细血管楔压升高,较闭塞前均具有统计学差异（P〈0．05）。实验过程中,体表心电图和冠状动脉内心电图均出现类似人AMI再灌注的心电图演变规律。结论选择性冠状动脉前降支急性闭塞、再灌注、微血栓注入制备的无复流小型猪动物模型是可行的。     

Preventive Role of Magnesium on Skeletal Muscle Ischemia–Reperfusion Injury—an Experimental Study

The present study aims to explore whether Mg infusion has a preventive effect on ischemia–reperfusion injury in rats. A total of 20 Sprague-Dawley-type adult male rats were used. In group 1 (control), 0.9% isotonic solution was administered. In group 2 (experiment), magnesium sulfate (0.5 mg per 100 g) was administered. Ischemia was induced for 15 min for the two groups. Magnesium (Mg), interleukin 8 (IL-8), and malondialdehyde levels were analyzed in blood, while edema, neutrophil infiltration, eosinophilia, loss of striation, and nucleolization were evaluated in histopathological examination. Mg levels in the experiment group were higher than those in the control group after ischemia–reperfusion (p < 0.05). In the control group, postischemia and postreperfusion IL-8 values were higher than preoperative values (p < 0.05). As for eosinophilia and loss of striation values, these were higher in the experiment group after ischemia–reperfusion than the values in the control group (p < 0.05). Histopathologically, Mg infusion cannot prevent the tissue injury triggered in ischemia–reperfusion periods. Eosinophilia can be one of the major and earliest markers of ischemia–reperfusion injury.     

Preconditioning with a Novel Metallopharmaceutical NO Donor in Anesthetized Rats Subjected to Brain Ischemia/Reperfusion

Rut-bpy is a novel nitrosyl–ruthenium complex releasing NO into the vascular system. We evaluated the effect of Rut-bpy (100 mg/kg) on a rat model of brain stroke. Forty rats were assigned to four groups (Saline solution [SS], Rut-bpy, SS+ischemia–reperfusion [SS+I/R] and Rut-bpy+ischemia–reperfusion [Rut-bpy+I/R]) with their mean arterial pressure (MAP) continuously monitored. The groups were submitted (SS+I/R and Rut-bpy+I/R) or not (SS and Rut-bpy) to incomplete global brain ischemia by occlusion of the common bilateral carotid arteries during 30 min followed by reperfusion for further 60 min. Thirty minutes before ischemia, rats were treated pairwise by intraperitoneal injection of saline solution or Rut-bpy. At the end of experiments, brain was removed for triphenyltetrazolium chloride staining in order to quantify the total ischemic area. In a subset of rats, hippocampus was obtained for histopathology scoring, nitrate and nitrite measurements, immunostaining and western blotting of the nuclear factor- κB (NF-κB). Rut-bpy pre-treatment decreased MAP variations during the transition from brain ischemia to reperfusion and decreased the fractional injury area. Rut-bpy pre-treatment reduced NF-κB hippocampal immunostaining and protein expression with improved histopathology scoring as compared to the untreated operated control. In conclusion, Rut-bpy improved the total brain infarction area and hippocampal neuronal viability in part by inhibiting NF-κB signaling and helped to stabilize the blood pressure during the transition from ischemia to reperfusion.     

The HMGB1–TLR4 axis contributes to myocardial ischemia/reperfusion injury via regulation of cardiomyocyte apoptosis

Toll-like receptor 4 (TLR4) and its ligand high mobility group box 1 (HMGB1), are known for playing central roles in ischemia–reperfusion injury in myocardium. However, the detailed mechanisms of TLR4 and HMGB1 are not fully understood. The aim of this study was to investigate the effects and possible mechanisms of the HMGB1–TLR4 axis and cardiomyocyte apoptosis on myocardial ischemic damage. Artificial oxygen ventilated anesthetized C3H/HeN mice and C3H/HeJ mice were subjected to 30 min of left anterior descending coronary artery occlusion followed by 6 h of reperfusion. The myocardial infarct size, HMGB1 levels, apoptosis index, Bax, Bcl-2 and TNF-α mRNA levels were assessed. The results showed that a lowered amount of cardiomyocyte apoptosis and infarct size in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency notably inhibited the expression of HMGB1 and TNF-a, both of which were up-regulated by ischemia/reperfusion. These findings suggest that the HMGB1–TLR4 axis plays a pathogenic role in triggering cardiomyocyte apoptosis during myocardial I/R injury and that the possible mechanism for this process is the result of released cytokines and inflammatory response involved in the HMGB1/TLR4-related pathway.     

Remote renal preconditioning-induced cardioprotection: a key role of hypoxia inducible factor-prolyl 4-hydroxylases

Remote preconditioning is a unique phenomenon in which brief episodes of ischemia and reperfusion to remote organ protect the target organ against sustained ischemia–reperfusion (I/R)-induced injury. Protective effects of remote renal preconditioning (RRPC) are well established in heart, but their mechanisms still remain to be elucidated. So, the present study was designed to investigate the possible role of oxygen-sensing hypoxia inducible factor-prolyl 4-hydroxylases (HIF-P4Hs) in RRPC-induced cardioprotection in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion and reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Ethyl 3,4-dihydroxybenzoate (EDHB) and α-ketoglutarate (α-KG) were employed as HIF-P4Hs inhibitor and activator, respectively. Diethyldithiocarbamic acid (DDCA) was employed as NFkB inhibitor. Remote renal preconditioning prevented I/R-induced myocardial injury and produced cardioprotective effects. Pharmacological preconditioning with EDHB (100 mg kg⁻¹ i.p.) mimicked the cardioprotective effects of RRPC. However, α-KG (200 mg kg⁻¹ i.p.) and DDCA (150 mg kg⁻¹ i.p.) abolished cardioprotective effects of RRPC and EDHB. So, it may be concluded that inhibition of HIF-P4H has a key role in RRPC-induced cardioprotection. Further, remote preconditioning-induced HIF-P4H inhibition may have triggered a transduction pathway involving activation of NFkB.     

Activation of Different Neuronal Phenotypes in the Rat Brain Induced by Liver Ischemia–Reperfusion Injury: Dual Fos/Neuropeptide Immunohistochemistry

The aim of the present study was to reveal the effect of liver ischemia–reperfusion injury (LIRI) on the activity of selected neuronal phenotypes in rat brain by applying dual Fos-oxytocin (OXY), vasopressin (AVP), tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), corticoliberine (CRH), and neuropeptide Y (NPY) immunohistochemistry. Two liver ischemia–reperfusion models were investigated: (i) single ligation of the hepatic artery (LIRIa) for 30 min and (ii) combined ligation of the portal triad (the common hepatic artery, portal vein, and common bile duct) (LIRIb) for 15 min. The animals were killed 90 min, 5 h, and 24 h after reperfusion. Intact and sham operated rats served as controls. As indicated by semiquantitative estimation, increases in the number of Fos-positive cells mainly occurred 90 min after both liver reperfusion injuries, including activation of AVP and OXY perikarya in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and TH, NPY, and PNMT perikarya in the catecholaminergic ventrolateral medullar A1/C1 area. Moreover, only PNMT perikarya located in the A1/C1 cell group exhibited increased Fos expression 5 h after LIRIb reperfusion. No or very low Fos expression was found 24 h after reperfusion in neuronal phenotypes studied. Our results show that both models of the LIRI activate, almost by the same effectiveness, a number of different neuronal phenotypes which stimulation may be associated with a complex of physiological responses induced by (1) surgery (NPY, TH, PNMT), (2) hemodynamic changes (AVP, OXY, TH, PNMT), (3) inflammation evoked by ischemia and subsequent reperfusion (TH), and (4) glucoprivation induced by fasting (NPY, PNMT, TH). All these events may contribute by different strength to the development of pathological alterations occurring during the liver ischemia–reperfusion injury.     

The Change of the Spinal Cord Ischemia–Reperfusion Injury in Mitochondrial Passway and the Effect of the <Emphasis Type="Italic">Ginkgo biloba</Emphasis> Extract’s Preconditioning Intervention

In order to explore whether the apoptosis in ischemia–reperfusion injury could be affected by Ginkgo biloba extract (GBE) and the free radical scavenger GBE could suppress this affection. Rabbits were randomly divided into sham group, ischemia group, ischemia–reperfusion group (1, 6, 24, 48 h), the drug group (1, 6, 24, 48 h). Measure the rate of apoptosis by flow cytometry, the caspase 9 and apoptosis-inducing factor (AIF) in the cytoplasm and serum by ELISA. Compared with the sham group and ischemia group, the reperfusion group increased the rate of apoptosis, the caspase 9 and AIF in serum have a peak at 24 h after reperfusion, in the cytoplasm the peak at 6 h.GBE inhibit performance has the systemic and local aspects. The apoptosis of nerve cells after spinal cord ischemia–reperfusion has the relationship with the mitochondrial caspase-dependent and caspase-independent pathways and both the local and systemic role. GBE inhibits nerve cell apoptosis by these ways.     

适用于Langendorff离体心脏灌流大鼠心肌梗死动物模型的建立

目的建立适用于Lansendorff离体心脏灌流大鼠心肌梗死的动物模型,为评价干细胞移植对急性心肌梗死后的心功能变化提供基础。方法选用Sprague-Dawley（SD）大鼠16只,结扎其左冠状动脉前降支中远1/3处,在结扎前后通过MPA多导生理记录仪连续描记心电图;4周后再次开胸进行Langendorff离体心脏灌流测定左室心功能和心肌组织病理学检查;另选仅开关胸后存活的10只SD大鼠作为对照组。结果造模成功率为62．50％（10／16）;心电图动态监测在冠脉结扎后出现ST-T抬高的融合波,30min后可见病理性Q波;4周后Langendorff离体心脏灌流装置系统检测显示左室收缩压峰值（LVSP）、左室内压等容相最大上升及下降速率（＋dp／dtmax,-dp/dtmax）等指标较对照组降低,左室舒张末压峰值（LVEDP）则反之;病理组织切片可见结扎区域心肌纤维排列紊乱、坏死心肌被纤维组织取代。结论通过结扎左冠脉前降支的方法,4周后能够形成稳定的适用于Langendorff离体心脏灌流的心肌梗死动物模型,该模型能应用于干细胞移植对心脏功能影响的研究。     

Neutrophil elastase inhibitor, sivelestat sodium hydrate prevents ischemia–reperfusion injury in the rat bladder

In the present study, we evaluated the effect of neutrophil elastase inhibitor, sivelestat sodium hydrate on ischemia–reperfusion injury in the rat bladder. Rat abdominal aorta was clamping with a small clip to induce ischemia–reperfusion injury in the bladder. Eight-week-old male Sprague Dawley rats were divided into four groups; sham-operated control rats, 30 min ischemia–60 min reperfusion (IR) rats, and IR rats treated with 15 or 60 mg/kg of sivelestat sodium hydrate. Sixty minutes prior to induction of ischemia, sivelestat sodium hydrate was administrated intraperitoneally. Real-time monitoring of blood flow and nitric oxide (NO) release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. The NO₂–NO₃ and malonaldehyde (MDA) concentrations were measured in the experimental urinary bladders. Clamping of the abdominal aorta, blood flow was rapidly decreased and NO release was gradually increased. After removing the clip, blood flow was rapidly increased and NO release was gradually returned to the basal level. These movements of blood flow and NO release were inhibited by treatment with sivelestat sodium hydrate in a dose-dependent manner. Both NO₂–NO₃ and MDA concentrations in the bladder were increased by induction of IR, and NO₂–NO₃ and MDA concentrations were decreased by treatment with high dose of sivelestat sodium hydrate significantly. Our data indicated that sivelestat sodium hydrate could inhibit increasing NO₂–NO₃ and MDA concentrations by IR, and it has potentiality protective effects on IR injury in the rat urinary bladder.     

Roles of mitochondrial dynamics modulators in cardiac ischaemia/reperfusion injury

The current therapeutic strategy for the management of acute myocardial infarction (AMI) is to return blood flow into the occluded coronary artery of the heart, a process defined as reperfusion. However, reperfusion itself can increase mortality rates in AMI patients because of cardiac tissue damage and dysfunction, which is termed ‘ischaemia/reperfusion (I/R) injury’. Mitochondria play an important role in myocardial I/R injury as disturbance of mitochondrial dynamics, especially excessive mitochondrial fission, is a predominant cause of cardiac dysfunction. Therefore, pharmacological intervention and therapeutic strategies which modulate the mitochondrial dynamics balance during I/R injury could exert great beneficial effects to the I/R heart. This review comprehensively summarizes and discusses the effects of mitochondrial fission inhibitors as well as mitochondrial fusion promoters on cardiac and mitochondrial function during myocardial I/R injury. The comparison of the effects of both compounds given at different time‐points during the course of I/R injury (i.e. prior to ischaemia, during ischaemia and at the reperfusion period) are also summarized and discussed. Finally, this review also details important information which may contribute to clinical practices using these drugs to improve the quality of life in AMI patients.     

T5 spinal cord transection increases susceptibility to reperfusion-induced ventricular tachycardia by enhancing sympathetic activity in conscious rats

We recently documented that paraplegia (T(5) spinal cord transection) alters cardiac electrophysiology and increases the susceptibility to ventricular tachyarrhythmias induced by programmed electrical stimulation. However, coronary artery occlusion is the leading cause of death in industrially developed countries and will be the major cause of death in the world by the year 2020. The majority of these deaths result from tachyarrhythmias that culminate in ventricular fibrillation. beta-Adrenergic receptor antagonists have been shown to reduce the incidence of sudden cardiac death. Therefore, we tested the hypothesis that chronic T(5) spinal cord transection increases the susceptibility to clinically relevant ischemia-reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity. Intact and chronic (4 wk after transection) T(5) spinal cord-transected (T(5)X) male rats were instrumented to record arterial pressure, body temperature, and ECG. In addition, a snare was placed around the left main coronary artery. The susceptibility to sustained ventricular tachycardia produced by 2.5 min of occlusion and reperfusion of the left main coronary artery was determined in conscious rats by pulling on the snare. Reperfusion culminated in sustained ventricular tachycardia in 100% of T(5)X rats (susceptible T(5)X, 10 of 10) and 0% of intact rats [susceptible intact, 0 of 10 (P < 0.05, T(5)X vs. intact)]. Beta-adrenergic receptor blockade prevented reperfusion-induced sustained ventricular tachycardia in T(5)X rats [susceptible T(5)X 0 of 8, 0% (P < 0.05)]. Thus paraplegia increases the susceptibility to reperfusion-induced sustained ventricular tachycardia due to enhanced sympathetic activity.     

Activation of mitochondrial μ-calpain increases AIF cleavage in cardiac mitochondria during ischemia-reperfusion

Ubiquitous calpains (calpain I and II) are generally recognized as cytosolic proteins. Recently, mitochondrial localized calpain I (μ-calpain) has been identified. Activation of mito-μ-calpain cleaves apoptosis inducing factor (AIF), a flavoprotein located within the mitochondrial intermembrane space, in liver mitochondria, but not in brain mitochondria. We first tested if activation of mito-μ-calpain cleaves AIF in isolated heart mitochondria. A decrease in AIF content within mitochondria increases cardiac injury during ischemia–reperfusion by augmenting oxidative stress. We hypothesize that the activation of mito-μ-calpain by calcium overload during ischemia–reperfusion results in decreased AIF content within mitochondria by cleaving AIF. The μ-calpain was present within mouse heart mitochondria, mostly in the intermembrane space. Exogenous calcium treatment induced a calpain-dependent decrease of mitochondrial AIF content in isolated mouse heart mitochondria. This process was blocked by a calpain inhibitor (MDL-28170). The Mitochondrial μ-calpain activity was increased by 160 ± 15% during ischemia–reperfusion compared to time control. In contrast, the mitochondrial AIF content was decreased by 52 ± 7% during reperfusion vs. time control in the buffer perfused mouse heart. Inhibition of mito-μ-calpain using MDL-28170 decreased cardiac injury by preserving AIF content within mitochondria during ischemia–reperfusion. Thus, activation of mito-μ-calpain is required to release AIF from cardiac mitochondria. Inhibition of calpains using MDL-28170 decreases cardiac injury by inhibiting both cytosolic calpains and mito-μ-calpain during ischemia–reperfusion.     

ATPase inhibitory factor 1 protects the heart from acute myocardial ischemia/reperfusion injury through activating AMPK signaling pathway

Rationale: Myocardial ischemia/reperfusion (I/R) injury is a common clinic scenario that occurs in the context of reperfusion therapy for acute myocardial infarction (AMI). The mitochondrial F1Fo-ATPase inhibitory factor 1 (IF1) blocks the reversal of the F1Fo-ATP synthase to prevent detrimental consumption of cellular ATP and associated demise. In the present study, we study the role and mechanism of IF1 in myocardial I/R injury.Methods: Mice were ligated the left anterior descending coronary artery to build the I/R model in vivo. Rat hearts were isolated and perfused with constant pressure according to Langendorff. Also, neonatal cardiomyocytes hypoxia-reoxygenation (H/R) model was also used. Myocardial infarction area, cardiac function, cellular function, and cell viability was conducted and compared.Results: Our data revealed that IF1 is upregulated in hearts after I/R and cardiomyocytes with hypoxia/re-oxygenation (H/R). IF1 delivered with adenovirus and adeno-associated virus serotype 9 (AAV9) ameliorated cardiac dysfunction and pathological development induced by I/R ex vivo and in vivo. Mechanistically, IF1 stimulates glucose uptake and glycolysis activity and stimulates AMPK activation during in vivo basal and I/R and in vitro OGD/R conditions, and activation of AMPK by IF1 is responsible for its cardioprotective effects against H/R-induced injury.Conclusions: These results suggest that increased IF1 in the I/R heart confer cardioprotective effects via activating AMPK signaling. Therefore, IF1 can be used as a potential therapeutic target for the treatment of pathological ischemic injury and heart failure.     

Bcl-xL gene transfer protects the heart against ischemia/reperfusion injury

Ischemia and reperfusion (I/R) injury causes the progression of cardiac dysfunction. The prevention of cardiomyocyte-loss due to I/R injury is important for the treatment of heart failure. Therefore, we employed antiapoptotic Bcl-xL protein to prevent I/R injury in the heart and evaluated the cardioprotective effect of Bcl-xL transduction by adenoviral vector (Adv) after I/R injury. Adv with Bcl-xL gene was injected in the rat heart 4 days prior to I/R. The prevention of cardiac performance-loss and the reduction of cardiac apoptosis, after 30min ischemia and 30min reperfusion of global I/R, were demonstrated in the heart with adenoviral Bcl-xL transduction. Also, significant reductions of the infarct size and serum creatine kinase levels were observed in the heart transduced with Bcl-xL gene compared with control after 30min ischemia and 24h reperfusion of the left anterior coronary artery. Thus, Bcl-xL may serve as a potential therapeutic tool for cardioprotection.     

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.