The effects of potassium concentration in reperfusion solution upon myocardial protection]

The effects of potassium in reperfusion solution (RS) and the influence of sodium on this effect were studied. Experimental time course was as followed: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas Cardioplegic Solution followed by global ischemia for 33 or 35 min at 37.5 degrees C, 15 min early Langendorff reperfusion with several different potassium concentration modified with Krebs Henseleit Bicarbonate Buffer (KHBB) containing 145 mM and 110 mM sodium and 5 min late reperfusion with KHBB, followed by 20 min working perfusion. Potassium in RS possessed bell shaped dose response nature with optimal concentration of 10 mM in the condition of 145 mM sodium but 6 m in the condition of 110 mM in terms of percent recovery of aortic flow. Although higher potassium reperfusion produced less Creatine Kinase leakage.     

The effects of magnesium concentration in reperfusion solution upon myocardial protection]

Experimental time course was as follows: 20 min working perfusion, 3 min cardioplegic infusion with St Thomas Cardioplegic Solution followed by global ischemia for 35 min at 37.5 degrees C, 15 min first Langendorff reperfusion with reperfusion solution (RS) with various concentrations of Mg and 5 min second reperfusion with KHBB, followed by 20 min working. Cardiac functions were measured during pre and post working perfusion and CK leakage were measured during reperfusion. Percent recoveries of aortic flow at the Mg concentration of 0, 0.6, 1.2, 3.0, 6.0, 12 mM were 21 +/- 5, 22 +/- 3, 48 +/- 2, 37 +/- 4, 28 +/- 3, 15 +/- 3 (%) (mean +/- SEM), respectively. Our data indicated that 1.2 mM Mg of RS possessed protective properties with bell shaped dose response characteristics.     

The effects of calcium concentration of reperfusion solution upon myocardial protection]

The effects of several calcium concentrations in reperfusion solution were studied. Experimental time course was as followed: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas cardioplegic solution (STS) followed by global ischemia for 30 min at 37.5 degrees C, 15 min early Langendorff reperfusion with reperfusion solution and 5 min late reperfusion with Krebs Henseleit bicarbonate buffer [( Calcium] = 2.5 mM), followed by 20 min working perfusion. Percent recoveries of aortic flow at the Ca concentration of 0, 0.1, 0.6, 1.2, 1.8, 2.5 mM were 0, 14 +/- 1, 43 +/- 4, 64 +/- 3, 55 +/- 2, 59 +/- 1 (%), respectively. Our data indicated that reperfusion solution with less than 1.2 mM calcium reduced the protective properties of STS.     

The effects of osmolarity in reperfusion solution upon myocardial protection]

The effects of several different osmolarity in reperfusion solution were studied. Experimental time course was as follows: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas Cardioplegic Solution (STS) followed by global ischemia for 33 min at 37.5 degrees C, 15 min early Langendorff reperfusion with different osmolarity by adding sucrose and 5 min late reperfusion with Krebs Henseleit bicarbonate buffer, followed by 20 min working perfusion. Percent recoveries of aortic flow showed that 290 mOsm/L in reperfusion solution possessed optimal protective properties with bell shaped dose response characteristics.     

Decreasing sarcoplasmic reticular calcium gives rise to myocardial protection? —The effect of thapsigargin for myocardial protection under conditions of normothermia—

Deceasing sarcoplasmic reticular (SR) calcium may contribute to the myocardiac protection against ischemia and reperfusion-induced injury. Therefore, using the isolated working rat heart model, we investigated the effect of Thapsigargin (TH)-induced SR calcium diminution on the myocardial protection when added either before onset of ischemia or at time of reperfusion under conditions of normothermic ischemia. Hearts (n=6/group) from male Wistar rats were aerobically (37°C) perfused (20 min) with bicarbonate buffer. In the experimental protocol A, this was followed by a 3 min infusion of St. Thomas’ Hospital cardioplegic solution No. 2 (STS) containing various concentrations of TH. Hearts were then subjected to 34 min of normothermic (37°C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). Reperfusion cardiac functions at 20 min of working perfusion was measured and compared with the preischemia values. STS added to 0.1 and 0.25 μmol/L TH improved recovery of aortic flow after 20 min reperfusion from 47 ± 3% in the TH free controls to 62 ± 3, 63 ± 2% (n=6) (p<0.05). There was no difference in creatine kinase (CK) leakage during Langendorff reperfusion between the TH treated groups and the control group. In the experimental protocol B, 3 min of cardioplegia without TH and 34 min of ischemia (37°C) were followed by a 10 min Langendorff reperfusion with various concentrations of TH, then 10 min Langendroff reperfusion for washing out, and 20 min working reperfusion. When TH was added to reperfusate the recovery of aortic flow did not change. 0.5 μmol/L TH group had the detelious effect. Thus, TH, when added to the cardioplegia, enhanced myocardial protection. We conclude that lessened uptake of Ca²⁺ into sarcoplasmic reticulum by inhibitors of the Ca²⁺-ATPase pump can decrease ischemia and reperfusion-induced injury.     

The effects of Na movement on surgical myocardial protection: the role of the Na+-H+ exchange system and Na-channel in the development of ischemia and reperfusion injury.

OBJECTIVES: We investigated whether the Na+-H+ exchange inhibitor, HOE642 (Hoe), and/or the Na channel blocker, mexiletine (Mex), enhance a cardioprotective effect on St. Thomas' Hospital cardioplegic solution (STS) to clarify the mechanism by which intracellular Na+ is accumulated after cardioplegic arrest. MATERIALS AND METHODS: Isolated working rat hearts were perfused with Krebs-Henseleit bicarbonate buffer (KHBB). The hearts were then arrested with STS and subjected to normothermic global ischemia (30 min). This was followed by Langendorff reperfusion (15 min) and then a working reperfusion (20 min). In study A, we added Hoe (5, 10, and 20 microM), Mex (70 microM), or a combination of Hoe (20 microM) and Mex (70 microM), to STS. In study B, we added Hoe (20 microM), Mex (70 microM), or a combination of Hoe (20 microM) and Mex (70 microM) to KHBB during the first 3 min of Langendorff reperfusion. RESULTS: In study A, the addition of Hoe (10 and 20 microM) to STS showed a significantly greater postischemic recovery of cardiac output compared to the control group [63.1+/-5.7% (10 microM), 62.7+/-4.7% (20 microM), and 55.5+/-4.6% (control), respectively]. The postischemic recovery of cardiac output was significantly greater in the group of the combined addition (Hoe and Mex) to STS than that in the control, 20 microM Hoe, 70 microM Mex groups [70.3+/-3.7 (Hoe and Mex), 55.5+/-4.6% (control), 62.7+/-4.7% (Hoe 20 microM), and 60.2+/-4.7% (Mex 70 microM), respectively]. The myocardial water content in the postischemic period was 565.1+/-29.1, 525.8+/-2.9, 509.4+/-19.6, and 532.2+/-20.1; it was 497.3+/-9.1 mL/100 g dry weight in the control; and 10 microM Hoe, 20 microM Hoe, and 70 microM Mex in the combined use groups. In study B, there was no significant difference in the postischemic recovery of cardiac output in all experimental groups. CONCLUSION: The combined use of the Na+-H+ exchange inhibitor and Na+ channel blocker during cardioplegia may achieve a superior cardioprotective effect on myocardial damage because of ischemia and reperfusion.     

The effects of protease inhibitor upon the ischemia-reperfusion injury]

The purpose of the study is to investigate the effects of protease inhibitor (Nafamostat mesilate: NM) upon myocardial protection. Hearts were subjected to 20 min working control perfusion followed by 3 min cardioplegic infusion with the St. Thomas Cardioplegic Solution (ST) contained various concentrations of NM, and global ischemia for 33 min at 37 degrees C (Exp. 1) or 150 min at 20 degrees C (Exp. 2). Hearts were then converted to Langendorff reperfusion (the leakage of Creatine Kinase (CK) and Cathepsin B (Cat-B) ware measured) and 20 min working reperfusion. Various concentrations of NM added during Langendorff reperfusion (Exp. 3). During working perfusion cardiac functions (aortic flow (AoF), coronary flow (CoF), heart rate (HR), aortic pressure (AoP)) were measured, and expressed as the percent recovery of pre-ischemic control value. Post-ischemic recovery of AoF (%AoF) showed the bell-shaped dose-response curve, and the optimal dose was 3 microM (Exp. 1) and 10 microM (Exp. 2) respectively. There was a significant (p < 0.05) increase of %AoF in optimal dose compared with that in controls (64.2 +/- 1.2% vs 52.3 +/- 2.5% in Exp. 1, 68.9 +/- 3.1% vs 54.1 +/- 1.4% in Exp. 2). These increase of functional recovery reflected in the values for CK and Cat-B leakage. The addition of NM in ST reduced CK and Cat-B leakage significantly in the concentration of 5 microM (in Exp. 1) and 10 microM (in Exp. 2) respectively. But the addition of NM in reperfusate did not reduced CK leakage significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repetitive brief ischemia: intermittent reperfusion during ischemia ameliorates the extent of injury in the perfused kidney

Acute renal failure commonly follows reduced renal perfusion or ischemia. Reperfusion is essential for recovery but can itself cause functional and structural injury to the kidney. The separate contributions of ischemia and of reperfusion were examined in the isolated perfused rat kidney. Three groups were studied: brief (5 min) ischemia, 20 min ischemia, and repetitive brief ischemia (4 periods of 5 min) with repetitive intervening reperfusion of 5 min. A control group had no intervention, the three ischemia groups were given a baseline perfusion of 30 min before intervention and all groups were perfused for a total of 80 min. In addition, the effects of exogenous *NO from sodium nitroprusside and xanthine oxidase inhibition by allopurinol were assessed in the repetitive brief ischemia-reperfusion model. Brief ischemia produced minimal morphological injury with near normal functional recovery. Repetitive brief ischemia-reperfusion caused less functional and morphological injury than an equivalent single period of ischemia (20 min) suggesting that intermittent reperfusion is less injurious than ischemia alone over the time course of study. Pretreatment with allopurinol improved renal function after repetitive brief ischemia-reperfusion compared with the allopurinol-untreated repetitive brief ischemia-reperfusion group. Similarly, sodium nitroprusside reduced renal vascular resistance but did not improve the glomerular filtration rate or sodium reabsorption in the repetitive brief ischemia-reperfusion model. Thus, these studies show that the duration of uninterrupted ischemia is more critical than reperfusion in determining the extent of renal ischemia-reperfusion injury and that allopurinol, in particular, counteracts the oxidative stress of reperfusion.     

吗啡预处理-后处理对大鼠离体心脏缺血再灌注损伤的影响

目的 探讨吗啡预处理-后处理对大鼠离体心脏缺血再灌注损伤的影响.方法 雄性SD大鼠,体重180～200 g,应用Langendorff体外灌流装置,采用全心停灌45 min、再灌注60 min的方法制备大鼠离体心脏缺血再灌注模型.取模型制备成功的心脏40个,随机分为5组(n=8):缺血再灌注组(IR组)、吗啡预处理组(M1组)、吗啡后处理组(M2组)、吗啡预处理-后处理组(M1+M2组)、5-羟葵酸(5-HD)混合吗啡后处理组(5-HD+M2组).M1组全心停灌前30 min灌注含3.0 μmol/L吗啡的K-H液20 min,随后灌注K-H液10 min.M2组再灌注即刻灌注含3.0 μmol/L吗啡的K-H液10 min,随后灌注正常K-H液50 min.5-HD+M1组再灌注即刻灌注含3.0 μmol/L吗啡+10-4nunol/L 5-HD的K-H液10 min,随后灌注正常K-H液50 min.于再灌注60 min时,测定心肌肌酸激酶(CK-MB)活性,计算心肌梗死区与缺血危险区的比值(IS/AAR).结果 与IR组相比,其余各组IS/AAR减少,CK-MB活性降低(P<0.05);与M2组比较,5-HD+M2组CK-MB活性及IS/AAR升高(P<0.05);M1组、M2组和M1+M2组上述指标比较差异无统计学意义(P>0.05).结论 吗啡预处理.后处理虽然可减轻大鼠离体心脏缺血冉灌注损伤,但是与单独应用时效果相似,其原因可能是两者单独应用减轻心脏缺血再灌注损伤的机制均与开放线粒体ATP敏感性钾通道有关.     

The consequences of asanguineous versus sanguineous reperfusion after long-term preservation of the heart

We have used the heterotopically transplanted rat heart to investigate whether the nature (sanguineous or asanguineous) of the initial period of reperfusion after hypothermic cardioplegic storage influences the postischaemic recovery of the heart. Excised rat hearts were arrested by infusion (1 min at 25 degrees C followed by 2 min at 7.5 degrees C) with the St Thomas' Hospital cardioplegic solution, subjected to 4 h of storage at 7.5 degrees C and heterotopically transplanted over a fixed period of 45 min. Reperfusion was then carried out for 80 min according to one of the following protocols: 60 or 20 min of blood perfusion in situ followed by excision, and 20 or 60 min of in vitro perfusion with crystalloid solution (Groups I and II, respectively) or immediate excision and 80 min of crystalloid perfusion (Group III). Intraventricular balloons were used to define pressure-volume relationships at the end of the 80 min period of reperfusion. Tissue samples were then taken for assessment of water content, adenosine triphosphate (ATP) and creatine phosphate (CP) content. Mean left ventricular developed pressure (at a loading volume of 110 microliters) was 92 +/- 6, 79 +/- 6 and 51 +/- 6 mmHg in Groups I, II and III, respectively. Left ventricular end-diastolic pressure was lower in the initial blood reperfusion groups (25 +/- 4 and 21 +/- 3 mmHg in Group I and II, respectively, compared with 37 +/- 5 mmHg in Group III).(ABSTRACT TRUNCATED AT 250 WORDS)     

吡那地尔超极化停搏对大鼠离体心脏缺血再灌注时p38MAPK表达的影响

目的 评价吡那地尔超极化停搏对大鼠离体心脏缺血再灌注时p38丝裂原活化蛋白激酶(p38MAPK)表达的影响.方法 成年雄性SD大鼠48只,体重250～300 g,采用随机数字表法,将大鼠随机分为6组(n=8):自然停搏组(A组)、St.Thomas组(B组)、吡那地尔超极化停搏组(C组)、5-羟葵酸(5-HD)组(D组)、HMR-1098组(E组)和5-HD+HMR-1098组(F组).采用Langendorff离体心脏灌注模型,K-H液平衡灌注15 min后,A组阻断主动脉,不予停搏液灌注,使其自然停搏;B组灌注St.Thomas停搏液;C组灌注吡那地尔超极化停搏液;D组、E组和F组K-H液平衡灌注10 min后,分别灌注含5-HD、HMR-1098、5-HD+ HMR-1098的K-H液5min,再灌注吡那地尔超级化停搏液.心脏停跳缺血60 min后,K-H液再灌注30 min.于平衡灌注15 min和再灌注20 min时记录冠脉流量(CF)、心率(HR)、左室发展压(LVDP)、左室收缩压(LVSP)和左室压力瞬时最大变化率(dp/dtmax);于再灌注30 min时取心肌组织,采用Western blot法测定心肌磷酸化p38MAPK和非磷酸化p38MAPK的表达.结果 与C组相比,A组、B组、D组、E组和F组再灌注20min时CF、HR、LVSP、LVDP及dp/dt/dymax降低,再灌注30 min时磷酸化p38MAPK表达下调,非磷酸化p38MAPK表达上调(P＜0.05);与E组相比,D组和F组再灌注20 min时CF、HR、LVSP、LVDP及dp/dtmax降低,再灌注30 min时磷酸化p38MAPK表达下调,非磷酸化p38MAPK表达上调(P＜0.05).结论 吡那地尔超极化停搏可改善大鼠离体缺血再灌注心脏功能,其机制与上调磷酸化p38MAPK表达,下调非磷酸化p38MAPK表达有关,而这种调控作用与线粒体ATP敏感性钾通道关系更密切.     

不同剂量左旋甲状腺素钠预处理对幼龄大鼠心肌缺血再灌注损伤的影响

目的 评价不同剂量左旋甲状腺素钠预处理对幼龄大鼠心肌缺血再灌注损伤的影响.方法 健康雌性 Wistar大鼠48只,日龄35 d,体重120～140 g,采用随机数字表法,将其随机分为6组(n=8):对照组(C组)、缺血再灌注组(I/R组)和不同剂量左旋甲状腺素钠预处理组(LS1-4组).C组和I/R组大鼠采用普通饲料喂养7 d;LS1-4组大鼠除了采用普通饲料喂养之外,每天胃内灌注左旋甲状腺素钠10、20、40和80 μg/100 g.第8天时,抽取外周静脉血样,测定血清甲状腺激素水平.采用Langendorff装置建立大鼠离体心脏缺血再灌注模型.C组K-H液持续灌注80 min;其余各组K-H液平衡灌注30 min,然后全心缺血20 min,恢复灌注30 min.于平衡灌注20 min和再灌注30 min时,记录HR、SP、左心室内压最大上升速率(+dp/dtmax)和左心室内压最大下降速率(-dp/dtmax),计算再灌注30min时HR、SP、+dp/dtmax和-dp/dtmax的恢复率.于平衡灌注10 min和再灌注15 min时,收集冠状动脉流出液2 ml,测定CK-MB的活性.再灌注30 min时,取心室肌组织,采用Western blot法检测心肌热休克蛋白70(HSPT0)的表达,采用RT-PCR法测定甲状腺激素受体(TR)亚型(TRα1、TRα2和TRβ1)mRNA以及肌球蛋白重链α和β(MHCα和MHCβ)mRNA的表达.结果 与C组相比,I/R组HR、SP和±dp/dtmax的恢复率降低,冠脉流出液CK-MB活性升高,心肌MHCα mRNA表达下调,LS1-4组SP和±dp/dtmax的恢复率降低,冠脉流出液CK-MB活性升高,心肌HSP70和MHCα mRNA表达上调,LS2-4组血清甲状腺激素水平升高,心肌TRα1 mRNA表达上调(P＜0.05).与I/R组相比,LS1-4组HR和±dp/dtmax的恢复率升高,心肌HSP70表达上调,MHCa mRNA表达上调,MHCβ mRNA表达下调,LS1-3组冠脉流出液CK-MB活性降低,LS2-4组血清甲状腺激素水平升高,心肌TRα1 mRNA表达上调(P＜0.05).LS1组、LS2组、LS3组和LS4组的甲状腺激素血清水平随左旋甲状腺素钠剂量的增加逐渐升高(P＜0.05).与LS1组和LS2组相比,LS3组和LS4组冠脉流出液CK-MB活性升高,心肌HSP70表达下调(P＜0.05).结论 10 μg/100 g左旋甲状腺素钠胃内灌注预处理可减轻大鼠心肌缺血再灌注损伤,且不会导致甲状腺激素水平升高,其机制可能与心肌HSP70和MHCα mRNA表达上调有关.

Abstract：

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.     

自噬在二氮嗪减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 评价自噬在二氮嗪减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 清洁级健康雄性SD大鼠32只,体重220～250 g,采用随机数字表法,将大鼠随机分为4组(n=8):缺血再灌注组(I/R组)、二氮嗪组(D组)、自噬抑制剂渥曼青霉素+二氮嗪组(WD组)和渥曼青霉素组(W组).WD组与W组腹腔注射渥曼青霉素15μg/kg,I/R组与D组给予等容量生理盐水,30 min后处死大鼠,制备Langendorff灌流模型,I/R组和w组灌注K-H液、D组和WD组灌注含二氮嗪100μmol/L的K-H液10 min,随后停灌20 min,再灌注30 min.于灌注二氮嗪前即刻、停灌前即刻、再灌注30 min时记录心率(HR)、左室舒张末期压(LVEDP)、左心室发展压(LVDP),于再灌注30 min时测定心肌组织SOD活性和MDA含量,检测自噬相关蛋白Beclin-1的表达,透射电镜下观察自噬小体形成情况.结果 与I/R组比较,D组LVDP和HR、心肌SOD活性和心肌Beclin-1表达水平升高,LVEDP和心肌MDA含量降低(P＜0.05),WD组和W组心肌Beclin-1表达水平降低(P＜0.05),其余指标差异无统计学意义(P＞0.05);与D组比较,WD组和W组LVDP和心肌Beclin-1表达水平降低,MDA含量升高,W组LVEDP升高,心肌SOD活性降低(P＜0.05).D组可见大量自噬小体,WD组与I/R组可见少量自噬小体,W组见极少量自噬小体.结论 自噬参与了二氮嗪减轻大鼠心肌缺血再灌注损伤的过程.

Abstract：

Objective To evaluate the role of autophagy in attenuation of myocardial ischemia-reperfusion (I/R) injury by diazoxide in the isolated rat heart.Methods Thirty-two male SD rats were randomly assigned into 4 groups ( n = 8 each) : I/R group, diazoxide group (group D), an inhibitor of autophagy wortmannin + diazoxide group (group WT＞) and wortmannin group (group W) . The animals were anesthetized with intraperitoneal pento-barbital sodium 40 mg/kg. Their hearts were excised and passively perfused in a Langendorff apparatus with an oxygenated (95% O2-5% CO2 ) K-H solution at 37 °C . The isolated hearts were made globally ischemic for 20 min followed by 30 min reperfusion. In I/R and W groups, the isolated hearts were perfused with K-H solution for 10 min before ischemia, while the isolated hearts were perfused with K-H solution containing diazoxide 100 /xmol/L for 10 min before ischemia in D and WD groups. The HR, left ventricular end-diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP) were recorded immediately before perfusion with diazoxide, immediately before the end of perfusion and at 30 min of reperfusion.Myocardial tissues were obtained at 30 min of reperfusion for determination of SOD activity, MDA content and autophagy-related protein Beclin-1 expression (by immunohistochemistry). The formation of autophagosomes was observed by transmission electron microscopy. ResultsCompared with group I/R, LVDP, HR, SOD activity and Beclin-1 expression were significantly increased at 30 min of reperfusion, while LVEDP and MDA content were significantly decreased at 30 min of reperfusion in group D(P＜0.05),and Beclin-1 expression was significantly decreased in WD and W groups(P＜0.05).Compared with group D, LVDP and Beclin-1 expression were significantly decreased, and MDA content was significantly increased in WD and W groups, and LVEDP was significantly increased, while SOD activity decreased in group W (P＜0.05). Microscopic examination showed that a large number of autophagosomes, a small number of autophagosomes and an extremely small number of autophagosomes were observed in D, WD and I/R groups respectively. Conclusion Autophagy is involved in attenuation of myocardial I/R injury by diazoxide in the isolated rat heart.     

Effect of initial reperfusion temperature on myocardial preservation

The effect of initial postischemic reperfusion temperature on myocardial preservation was studied in the isolated working rat heart model. After baseline measurement of aortic flow rate, coronary flow rate, and heart rate, 40 hearts were subjected to 60 minutes of ischemic arrest at 15 degrees C induced with a single dose of cold potassium cardioplegic solution. Hearts were then revived with a 10 minute period of nonworking reperfusion at 28 degrees, 31 degrees, 34 degrees, or 37 degrees C (10 hearts each), followed by 5 minutes of nonworking reperfusion at normothermia, followed by 30 minutes of working perfusion. Repeat measurements of function were obtained and postischemic release of creatine kinase into coronary effluent was determined. Recovery of aortic flow was significantly reduced at lower initial reperfusion temperatures (75% at 28 degrees C versus 88% at 37 degrees C) and the effect was approximately linear throughout the range studied (p less than 0.05). Release of creatine kinase into coronary effluent was greater at lower initial reperfusion temperatures (421 ImU/min/gm wet weight at 28 degrees C versus 115 ImU/min/gm wet weight at 37 degrees C), also in a linear manner (p less than 0.05). In this model, initial postischemic hypothermic reperfusion is deleterious to cellular integrity and functional recovery of the preserved myocardium. Studies in higher animals and humans are warranted to further evaluate the effect of initial reperfusion temperature on myocardial preservation.     

Harmful effects of inotropic agents on myocardial protection.

Using an isolated working rat heart model, the pretreatment effects of positive inotropic agents on ischemia-reperfusion injury were investigated. The experiment consisted of (1) working control perfusion; (2) working perfusion with isoproterenol (I), milrinone (M), a combination of these drugs (I + M) and dibutyl-cyclic adenosine monophosphate (DB) followed by ischemic arrest for 33 minutes at 37 degrees C or 150 minutes at 20 degrees C and Langendorff reperfusion; and (3) working perfusion. Under conditions of normothermic ischemia, percent recoveries of postischemic cardiac output (mean +/- standard error of the mean) in the I, M, I + M, and DB groups were 37.8% +/- 12.7%, 61.3% +/- 3.1%, 0%, and 53.1% +/- 5.2%, respectively. Under conditions of hypothermic ischemia, the percent recoveries in I + M and DB groups were 10.9% +/- 7.9% and 29.8% +/- 9.5%; they were all significantly lower than that in the control group. The addition of diltiazem or ryanodine at several concentrations and lowering of the Ca2+ concentration in the St. Thomas' cardioplegic solution did not prevent I + M-induced injury. Our data suggest that pretreatment by I + M aggravated ischemia-reperfusion injury, and adjustments in Ca2+ concentration were not sufficient to prevent that injury.     

Isolated hyperthermic liver perfusion with high dose tumor necrosis factor alpha in pigs: an experimental study in preparation of clinical Use

Isolated hyperthermic perfusion of the liver was performed for 45 min in 27 pigs via hepatic artery and portal vein at mean inflow temperatures between 40.7 and 41.2 degrees C. In two study groups B and C (n = 9 pigs each) 50 microg recombinant human tumor necrosis factor-alpha (rhTNFalpha) per kg body weight were added to the perfusate, whereas in a control group A liver perfusion was done without rhTNFalpha. Before reperfusion the livers were washed out with Ringer's solution in all groups followed by a protein solution in group C. At 30 and 60 min after reperfusion the maximum systemic rhTNFalpha concentrations were significantly higher in group B with 68 and 61 ng/ml compared to 14.5 and 14.9 ng/ml in group C (p < 0. 05). Mean systemic porcine TNFalpha concentration was significantly higher in group B (217 pg/ml) compared to group C (50 pg/ml) 30 min after reperfusion (p = 0.012). Survival was 7/9 in group A and C and only 2/9 in group B with 6/7 pigs dying due to severe cardiopulmonary failure within 12 h after operation. In surviving pigs of group A and C only mild and transient hepatotoxicity was registered. The presented study underlines the feasibility of high dose rhTNFalpha application in an isolated hyperthermic liver perfusion system. Washout of the liver with a protein solution before reperfusion reduces systemic TNFalpha levels as well as associated lethal cardiocirculatory and hepatotoxic side effects.     

The role of magnesium in the endothelial dysfunction caused by global ischemia followed by reperfusion: in vitro study of canine coronary arteries

OBJECTIVE: To study the role of magnesium in the endothelial dysfunction of canine coronary arteries caused by cardiopulmonary bypass (CPB) global ischemia followed by reperfusion. DESIGN: Segments of canine coronary arteries were suspended in organ chambers to measure isometric contraction by prostaglandin F (2alpha), and relaxed by acetylcholine (ACh), sodium fluoride (NaF), calcium ionophore (A23187) and sodium nitroprusside (SNP) in crescent concentrations. The investigation protocol had groups with six dogs: CONTROL group (without CPB), CPB group (105 min of CPB without aortic cross-clamping), ISCH group (45 min of CPB with aortic cross-clamping), ISCH/REP group (45 min of aortic cross-clamping followed by 60 min of reperfusion). The coronary relaxations were evaluated with (phase I), without (phase II) and restored magnesium (phase III) to the organ bath. RESULTS: The presence of magnesium in the organ bath was associated with the greater relaxation in response to agonists of the nitric oxide production. The removal of magnesium from the organ bath was associated with the reduction in the intensity of vessel relaxation. The magnesium restoration to the organ bath was associated with the additional reduction in the intensity of relaxation with the exception of NaF that allowed re-acquisition of the relaxation observed in the presence of magnesium. CONCLUSION: This in vitro study demonstrates that magnesium ion favorably influences the nitric oxide production by the coronary endothelium, attenuating the endothelial dysfunction caused by global ischemia followed by reperfusion.     

Studies on optimal reperfusate temperature following hypothermic ischemic arrest in the isolated rat hearts

This study was undertaken to evaluate the optimal reperfusate temperature during early reperfusion period for 10 min of non-working mode followed by 20 min of normothermic working mode in the isolated hypothermic ischemic (20 degrees C, 60 min) rat hearts. The reperfusate temperature was selected 20 degrees C (group A) and 30 degrees C (group B) as a hypothermic reperfusion, and 37 degrees C (group C) for normothermia. Functional recovery of aortic flow rate (AFR), heart rate (HR), peak systolic pressure (PSP), coronary flow (CF), and rate pressure product was compared among three groups as well as myocardial biochemical (adenosine triphosphate (ATP), creatine phosphate (CP)) and ultrastructural change. During non-working reperfusion, HR and CF showed significant positive correlation to reperfusate temperature. At the end of 10 min of non-working reperfusion, myocardial creatine phosphate (CP) significantly increased in all groups beyond the pre-ischemic level, however CP in the group A and B was significantly better compared to that in the group C. And myocardial ATP was better recovered near to the pre-ischemic level in the hypothermic groups compared to the group C without significant recovery, being significantly different between group A and C. The integrity of mitochondrial ultrastructure was also well maintained in group A compared to group C. At the end of normothermic reperfusion in the working mode for 20 min, percent recovery of AFR was 85.1% (B), 73.3% (A) and 70.3% (C), and percent recovery of rate pressure product was 92.5% (A), 89.1% (B) and 78.6% (C) with significant difference between the hypothermic and the normothermic groups. PSP, CF and HR recovered without significant difference among three groups. Myocardial ATP and CP declined significantly during 20 min of working reperfusion to the same level as those of three groups. CP only in group C was significantly lower than pre-ischemic level, although ATP in all groups at the end of reperfusion was significantly lower than the pre-ischemic level. These data suggest that hypothermic (20 degrees C, 30 degrees C) reperfusion seemed to provide better functional and metabolic recovery in association with better preservation of ultrastructure through improving energy production-utilization balance in early reperfusion period. And rapid warming in the reperfusion period may deteriorate or delay the myocardial recovery from ischemic isult.     

Pulmonary artery occlusion and reperfusion causes microvascular constriction in the rabbit lung

INTRODUCTION: Reperfusion injury of the lung after ischemia is associated with altered alveolar blood flow and ventilation-perfusion mismatch, which is a significant cause of morbidity and mortality after lung transplantation. We examined the effect of ischemia and reperfusion on the tone of individual subpleural arterioles in the pulmonary circulation by using video microscopy with polarized epiillumination. METHODS: In 11 open-chested rabbits anesthetized with pentobarbital (2.3 to 2.5 kg), we ventilated the lungs through the lower trachea (air or 50% oxygen) and examined the response of subpleural arterioles (diameter 75 +/- 13 microm) to ischemia (76 +/- 32 min) of the right lung caused by occluding the right main pulmonary artery. Observations were made during baseline, occlusion, and during early (20 to 32 min) and late (48 to 63 min) reperfusion using a long working distance lens (550x) with a dipping cone held at the pleural surface while the lungs were statically inflated (10 cm H2O) with oxygen for brief periods. Data are expressed as mean +/- standard deviation. RESULTS: Arteriolar diameter was decreased 57% +/- 19% during early reperfusion. There was a decrease in blood flow and alveolar walls were pale, indicating reduced capillary perfusion. During late reperfusion, arteriolar diameter was diminished (19% +/- 26%); flow was still reduced. Overall pulmonary vascular resistance increased during early reperfusion but returned to baseline level during late reperfusion. Arterial partial pressure of oxygen averaged 200 mm Hg during ischemia and reperfusion. CONCLUSIONS: Constriction of small arterioles by ischemia and reperfusion can have a significant effect on the early phase of ventilation-perfusion mismatch and pulmonary dysfunction by altering alveolar perfusion. This response does not appear to be mediated by hypoxia because it was not prevented by ventilation with oxygen.     

Comparison of pulsatile and nonpulsatile perfusion of the lung in an extracorporeal large animal model

OBJECTIVE: Extracorporeal lung-perfusion models are widely used to evaluate pulmonary preservation techniques and reperfusion injury. However, these models mainly depend on nonpulsatile flow, which is not physiological and can subsequently lead to pulmonary edema. Observation in a standardized setting and reliability of functional and structural data assessment are therefore limited. To overcome these limitations we developed a new extracorporeal large animal lung perfusion model utilizing pulsatile flow to perfuse the pulmonary vasculature. METHODS: Lungs of juvenile domestic pigs were in situ preserved with 2 liters Perfadex and stored for 3 h at 10 degrees C. Thereafter, reperfusion of the lung was performed in an extracorporeal blood perfusion circuit employing either a modified roller pump with pulsatile module (300 ml/min; pulsation rate 90/min) or a standardized roller pump with continuous flow (30 ml/min). Ventilation was performed with physiologic room air (350 ml; 16/min) for 1 h. Pulsatile and nonpulsatile perfusion was performed in 2 groups (group NP: nonpulsatile; group P: pulsatile flow, n = 7) during reperfusion. Peak inspiratory pressure (PIP), mean pulmonary artery pressure (PAP), and oxygenation capacity (DeltaPO(2)) were continuously measured. For control of the effectiveness of the pulsatile perfusion pressure waveforms were obtained directly from the native pulmonary artery of both groups. Malondialdehyde (MDA) as a parameter for lipid peroxidation and endothelial cell damage was assessed at 10, 30 and 50 min reperfusion. At the end of the study, pulmonary water content was assessed by means of wet-to-dry ratio (W/D ratio). The tissue was further processed for microscopic analysis. RESULTS: PIP increased significantly in both groups during reperfusion. Mean PAP in both groups increased to 60 mm Hg after 20 min followed by a decrease after 60 min to 40 mm Hg. Pressure waveforms of the pulmonary artery showed sufficient pulsatility in the pulmonary vasculature with a systolic/diastolic pressure difference of 15 mm Hg whereas the pressure difference was 3-5 mm Hg in the nonpulsatile group. DeltaPO(2) was stable in groups NP and P during reperfusion (30 min: NP: 66.4 (62.2-88) mm Hg; P: 74.8 (65-81.7) mm Hg) without any statistically significant differences between the groups. MDA in group NP decreased over the reperfusion period from 6.2 (3.3-6.3) microM at 10 min to 5.2 (3.2-6.1) microM at 50 min, whereas in group P the level increased and was significantly higher after 50 min reperfusion compared to group NP [6.6 (6.1-9.2) microM at 50 min; p = 0.016]. W/D ratio was 6.7 (6.3-7.0) in group NP and 6.8 (6.3-7.6) in group P. Light microscopy evaluation showed no differences between both groups regarding severity of intra-alveolar and interstitial edema and numbers of intra-alveolar, intracapillary and interstitial granulocytes. CONCLUSION: Although effective pulsatile perfusion of the pulmonary vasculature was achieved by means of a modified roller pump, this measure obviously did not improve functional parameters nor did it significantly reduce the edema formation after 3 h ischemia in this extracorporeal lung perfusion model. The use of pulsatile perfusion is therefore not mandatory in the extracorporeal setting of a large animal lung perfusion model.