Bretschneider's Solution Research Articles

Necrostatin-1 inhibits Hmgb1-IL-23/IL-17 pathway and attenuates cardiac ischemia reperfusion injury.

Ischemia reperfusion (IR) injury is a major issue in cardiac transplantation and inflammatory processes play a major role in myocardial IR injury. Necrostatin-1 (Nec-1) is a small molecule capable of inhibiting RIP1 kinase activity and attenuates inflammation-mediated tissue injury. In our study, hearts of C57Bl/6 mice were flushed and stored in cold Bretschneider solution for 8h and then transplanted into syngeneic recipients. We found that Nec-1 decreased cardiomyocyte necrosis and recruitment of neutrophils and macrophages. Troponin T (TnT) production on 24h after myocardial IR injury was reduced by Nec-1 administration. Cardiac output at 60mmHg of afterload pressure was significantly increased in hearts with Nec-1 administration and the cardiac allograft survival in Nec-1-treated animals was significantly prolonged (MST=90days in IR+Nec-1 group, P<0.05 as compared with IR group, MST=83.5days). Nec-1 treatment attenuated ROS generation and increased expression of NOS2 and COX-2. The expression of Hmgb1, IL-23, and IL-17A were also decreased with Nec-1 administration. Furthermore, the decreased TnT expression induced by Nec-1 was abrogated with exogenous Hmgb1 administration. In conclusion, Nec-1 played a protective role in cardiomyocyte IR injury, and this was associated with inhibited Hmgb1-IL-23/IL-17 pathway.

Myocardial Protection in Donor Heart Preservation: A Comparison Between Bretschneider's Histidine–Tryptophan–Ketoglutarate Solution and Cold Blood Cardioplegia

BackgroundOptimal myocardial protection for donated hearts is crucial to improve outcomes of heart transplantation and reduce morbidity and mortality. This study aimed to compare the efficacy of myocardial protection using single dose of Bretschneider's histidine–tryptophan–ketoglutarate (HTK) solution and repeated doses of cold blood cardioplegia (CBC) in donor heart preservation. MethodsSixty-seven patients undergoing heart transplantation in Tri-Service General Hospital, Taipei, Taiwan between 2002 and 2012 were enrolled in this study. Patients were divided into an HTK group and a CBC group based on the preservation solution used to protect the donated hearts. The perioperative variables and postoperative outcomes were retrospectively reviewed. ResultsThere were no statistic differences about demographic data in donors and recipients between the 2 groups. There were no significant differences in postoperative cardiac enzymes, hemodynamic data, length of stay in intensive care, or 30-day mortality between the groups. The HTK group showed a trend of shorter pumping time (P = .091). Multivariate analyses reveal that the HTK group had higher postoperative inotropic score (P < .001) and shorter pumping time (P = .02). ConclusionsSingle dose of Bretschneider's HTK solution could effectively reduce pumping time and afford similar myocardial protection compared with repeated doses of CBC in the preservation of donated hearts.

RNase A in (Xeno)Transplantation

BackgroundCell injury, particularly as consequence of ischemia/reperfusion or acute rejection after (xeno)transplantation, leads to release of intracellular components like RNA. Extracellular RNA, (i) causes edema as a result of increasing permeability of blood vessels [1], (ii) is described as a procoagulation factor by activation of the contact phase system leading to thrombus formation and vessel occlusion [2], and (iii) promotes leukocyte recruitment to the vascular system by mobilising proinflammatory cytokines [3], and thus, triggers immune response. In rodent models of stroke and thrombosis, treatment with counteracting RNase A, a ribonuclease that specifically degrades single‐stranded RNA, was shown to result in less edema formation and vessel occlusion [4]. In search of additive drugs to protect (xeno)grafts from dysfunction, we investigated if treatment with RNase A in a rat model of heterotopic heart transplantation results in less edema and less coronary occlusion, and, therefore, in improved graft survival.MethodsBrown Norway rat cardiac allografts were transplanted into the abdomen of Lewis rats after perfusion with Bretschneider cardioplegic solution. Microvascular technique for aorto‐aortic anastomosis and pulmonary artery to inferior vena cava anastomosis (Langendorff) was used. Recipients were intravenously treated directly before transplantation and every other day with RNase A (50 μg/kg) or vehicle (saline, n = 6 in each group). In addition, in six cases the donor graft was perfused with Bretschneider solution containing RNase A (8 μg in 10 ml), and afterwards transplanted into RNase A treated recipients. The primary end point of the study was graft survival, which was determined by daily examination of the graft function by palpation and in case of inconclusiveness by echocardiography. A tolerance study was performed treating animals daily with 50 μg/kg of RNase A, with 1,000 μg/kg of RNase A, or vehicle, respectively, for 28 days (n = 3 in each group). All explanted hearts were collected for histological evaluation.ResultsMean graft survival was 6.5 ± 0.55 day (range 6–7 day) in vehicle treated animals vs. 10.2 ± 1.0 day (range 9–11 day) in RNase A treated animals (P = 0.002). Directly after explantation donor grafts had a mean weight of 0.94 ± 0.03 g (range 0.89–0.98 g). After been rejected, donor graft weight increased to 2.06 ± 0.52 g (range 1.59–2.77 g) in vehicle treated recipients, in contrast to 1.28 ± 0.27 g (1.05–1.61 g) in RNase A treated recipients (P = 0.017). No supplemental benefit was observed by additive using of RNase A in Bretschneider solution (graft survival 9.50 ± 1.52 day, graft weight 1.38 ± 0.15 g) compared to single treatment of the recipient (graft survival P = 0.394, graft weight P = 0.589). Tolerance studies showed neither macroscopic nor histological differences compared to saline treated animals.Outlook: Further histological analyses will help to clarify if RNase A will keep the promise to reduce edema formation even in the field of transplantation, as suggested by the significant less graft weight in RNase A treated recipients. Moreover, we will investigate, if RNase A prevents leukocyte recruitment in the context of rejection reaction. Therefore, grafts of RNase A treated and untreated rats will be explanted on day 4 after transplantation.ConclusionsRNase A significantly improved graft survival. On the basis of these sweeping results, however, we suppose that RNase A could be an important adjuvant drug not only in allotransplantation but even in xenotransplantation.

Netrin-1 Attenuates Cardiac Ischemia Reperfusion Injury and Generates Alternatively Activated Macrophages

Ischemia reperfusion (IR) injury is a major issue in cardiac transplantation and inflammatory processes play a major role in myocardial IR injury. Netrin-1 is a laminin-related protein identified as a neuronal guidance cue and netrin-1 expressed outside the nervous system inhibits migration of leukocytes in vitro and in vivo and attenuates inflammation-mediated tissue injury. In our study, hearts of C57BL/6 mice were flushed and stored in cold Bretschneider solution for 8 h and then transplanted into syngeneic recipient. We found that netrin-1 decreased cardiomyocyte apoptosis and recruitment of neutrophils and macrophages. Troponin T (TnT) production on 24 h after myocardial IR injury was reduced by netrin-1 administration. Cardiac output at 60 mmHg of afterload pressure was significantly increased in hearts with netrin-1 administration (IR + Netrin-1: 59.9 ± 5.78 ml/min; IR: 26.2 ± 4.3 ml/min; P < 0.05). Netrin-1 treatment increased expression of the alternatively activated macrophage (AAM) markers arginase-1 (Arg-1) and mannose receptor (MR) and promoted proliferator-activated receptor γ (PPARγ) expression in cardiac allograft. Furthermore, decreased TnT expression and reduced allograft infiltration of neutrophils and monocytes/macrophages by netrin-1 was abolished with addition of PPARγ antagonist. In conclusion, netrin-1 attenuates cardiac IR injury and generates AAM which contributes to the protective effect of netrin-1.

Long pentraxin PTX3 attenuates ischemia reperfusion injury in a cardiac transplantation model

Ischemia reperfusion (IR) injury is a major issue in cardiac transplantation, and inflammatory processes play a major role in myocardial IR injury. Long pentraxin-3 (PTX3) is a member of a phylogenetically conserved group of acute-phase reactants that are involved in inflammation and innate immunity. In our study, hearts of C57Bl/6 mice were flushed and stored in cold Bretschneider solution for 8 h and then transplanted into syngeneic recipient. We found that both mRNA and protein levels of PTX3 were increased following myocardial IR injury; neutralizing antibody against PTX3 aggravated cardiomyocyte apoptosis and recruitment of neutrophils and macrophages. Troponin T (TnT) production on 24 h after myocardial IR injury was reduced by exogenous PTX3 administration and increased by PTX3 neutralization in comparison with control. Cardiac output at 60 mmHg of afterload pressure was also increased in hearts with exogenous PTX3 administration and decreased with PTX3 neutralization (PTX3: 58.4 ± 7.4 ml/min; 24.5 ± 3.8 ml/min; Anti-PTX3: 11.6 ± 1.7 ml/min; P < 0.05). Furthermore, PTX3 restricted expansion of γδ T cell that was the major source of IL-17A and down-regulated expression of IL-23 and IL-17A. In conclusion, PTX3 played a protective role in cardiomyocyte IR injury. PTX3 ameliorated cardiomyocyte apoptosis and infiltration of neutrophil and macrophage and then improved hemodynamic performance. This was associated with restricted γδ T-cell expansion and decreased IL-23/IL-17A expression.

Hmgb1-TLR4-IL-23-IL-17A Axis Promote Ischemia-Reperfusion Injury in a Cardiac Transplantation Model

Cardiac transplantation is the last resort for patients with end-stage heart failure. Ischemia-reperfusion (IR) injury is a major issue in cardiac transplantation. Inflammatory processes play a major role in myocardial IR injury. However, the cellular and molecular immune mechanisms of myocardial IR injury remain elusive. Hearts of C57BL/6 mice were flushed and stored in cold Bretschneider solution for 8 hr and then transplanted into syngeneic recipient. The involvement of high-mobility group box 1 (Hmgb1) and interleukin (IL)-17A was assessed in functional assays by neutralizing Hmgb1 or IL-17A. IL-17A was elevated after myocardial IR injury in cardiac transplantation. IL-17A was predominantly produced by γδT cells rather than CD4 or CD8 T cells infiltrated into the cardiac isografts. Neutralizing antibody against IL-17A or γδTCR attenuated cardiomyocyte apoptosis and neutrophil recruitment. Furthermore, a neutralizing IL-23p19 antibody decreased the level of IL-17A and neutrophil infiltration. Importantly, IL-23 and IL-17A were reduced after inhibition of macrophages and could not be induced in TLR4 mice after IR injury. Meanwhile, Hmgb1 increased after IR injury and the Hmgb1 inhibitor glycyrrhizin markedly reduced the production of IL-23 and IL-17A and ameliorated myocardial IR injury. The Hmgb1-TLR4-IL-23-IL-17A axis contributes to cardiomyocyte apoptosis, neutrophil accumulation and IR injury in cardiac transplantation.

56. Evaluation of a new solution for hypothermic machine perfusion (HMP) of the liver. II – A study in the perfused rat liver in vitro

The present study was undertaken to develop and investigate the possible benefit of a new solution for hypothermic machine perfusion (HMP) of livers for transplants. The portal vein and bile duct of Wistar rats were cannulated, the liver excised and connected on a recirculating perfusion system with 250 mL of BES-Gluconate-Polyethylene Glycol based solution (BGP-HMP) or with Bretschneider solution (HTK) at 5 °C. HMP was performed up to 24 h at a constant pressure of 40 mmH 2 O (equivalent to 25% of the normothermic portal pressure). Both perfusion solutions were air equilibrated during the HMP. After 24 h of HMP, the livers were reperfused at 37 °C with Krebs–Henseleit Dextran solution (Balaban, C.L., 2011. Artif Organs 35(5), 508–515). In both procedures, portal pressure and flow rate were measured and the intrahepatic resistance (IR) was calculated. Perfusate pH oscillations and enzymes (LDH, AST, and ALT) activities were evaluated during HMP and normothermic reperfusion. Also, the O 2 consumption, glucose, urea production and bile flow were measured during the normothermic reperfusion period. Liver perfusion variables and injury parameters during HMP Statistical differences in enzymes release ( p n = 5) during HMP. Enzyme activities in perfusate was (U/L.gliver): LDH, 10.01 ± 1.22 (HTK), 6.20 ± 1.03 (BGP–HMP); AST, 3.63 ± 0.62 (HTK), 1.25 ± 0.13 (BGP–HMP). In contrast, for ALT release no statistical differences were found. Liver function and viability during normothermic reperfusion The portal flow was 1.63 ± 0.41 vs. 2.85 ± 0.59 (mL/min.gliver) with HTK and BGP–HMP respectively. The IR and enzyme release after 90 min of reperfusion showed statistical differences between both groups. HTK solution showed higher values than BGP–HMP: IR (mmHg.min.gliver/mL) 4.96 ± 1.15 vs. 2.79 ± 0.66; LDH 43.04 ± 13.2 vs. 15.1 ± 5.43; AST 8.5 ± 2.7 vs. 3.46 ± 1.02; ALT 3.72 ± 1 vs. 1.04 ± 0.5. These results indicate that BGP–HMP is able to maintain better membrane integrity of the hepatocytes and reduce the hypothermic preservation and reperfusion injuries of the livers. Also, hematoxylin/eosin stain analysis showed better integrity in livers treated with BGP–HMP than livers perfused with HTK solution. With regard to bile flow and O 2 consumption, preserved livers with either solutions were able to produce bile, but the livers stored with BGP–HMP were able to uptake more O 2 (μmol/min.gliver) than livers preserved with HTK: 1.08 ± 0.08 vs. 0.65 ± 0.1. So, in the setted conditions, the livers are able to keep an active metabolism with both preservation solutions, but the BGP–HMP solution enhances the graft O 2 consumption capacity and reduces IR.

55. Evaluation of a new solution for hypothermic machine perfusion (HMP) of the liver. I – Composition and physicochemical parameters

There are many experimental as well as clinical reports indicating that HMP of marginal donor livers are superior to cold static preservation. However, there are still doubts as to the best perfusion preservation solution to use and the role of oxygenation during the HMP. For these reasons, we have developed a BES – Gluconate – Polyethylene Glycol based solution (BGP–HMP). This solution is used to protect the graft providing an appropriate oxygen concentration and by maintaining pH and vascular oncotic control during the HMP. The additions of substrates to decrease the ischemia reperfusion injury were also considered. Material and methods: BGP-HMP solution composition (mM) was; 100 Na + gluconate, 7 K + gluconate, 20 sucrose, 30 BES, 2.5 KH 2 PO4, 0.03 polyethylene glycol 35 kDA, 5 MgSO 4 , 3 glutathione, 5 adenosine, 15 glycine, 0.25 mg/mL streptomycin and 10 (U/mL) penicillin G. Osm: 297 ± 4 m Osm/kg H 2 O, pH = 7.40, Na + = 120 ± 2 and K + = 10 ± 1 mEq/L. The freezing point and the buffering capacity (BC) and 5 and 10 °C of the BGP-HMP solution were determined (Mamprin, M.E., 2008. Cryoletters 29, 121–133) and compared with HTK (Bretschneider solution) as the reference solution. The solubility curves of O 2 in BGP–HMP and HKT solutions were determined (Llarrull, M.S., 2007. Cryoletters 28, 313–328). The O 2 consumption of isolated rat livers perfused at 5 and 10 °C with BGP–HMP or HTK solutions saturated with air were also measured (Rodriguez, J.V., 2009. Cryoletters 30, 335–346). Results: the specific freezing point of the BGP–HMP sol. was −0.55 ± 0.05 °C and the BC were: BGP–HMP 15.44 and HTK 59 mEqH + /UpH/l solution respectively, there were no differences between 5 and 10 °C. The O 2 solubility at 5 and 10 °C and 760 mmHg was 351 and 314 μM O 2 for BGP–HMP and 370 and 325 μM O 2 for HTK. The O 2 consumption of rat livers after 5 h of perfusion at 5 and 10 °C with BGP–HMP was: 33 ± 5 and 61 ± 12, ( n = 4) and with HTK 38 ± 7 and 57 ± 4, ( n = 4) nmol O 2 /min/g.liver respectively. The livers were perfused at constant pressure (70 ± 10 mmH 2 O) and the perfusion flow was 0.15 ± 0.02 mL/min/g.liver. The O 2 extraction by the hypothermic perfused livers was 0.46 ± 0.16 and 0.67 ± 0.17 for BGP–HMP and 0.36 ± 0.01 and 0.45 ± 0.03 for HTK solution at 5 and 10 °C, respectively. Control livers perfused with Krebs–Henseleit–Dextran solution at 37 °C consume1150 ± 10 nmol O 2 /min/g.liver ( n = 3), the extraction ratio was 0.86 ± 0.03. Conclusion: we have included three key components in the BGP–HMP solution, gluconate as a non-diffusible anion, the aminosulfonic acid BES to improve the BC and PEG 35000 to provide vascular oncotic support. The BC of our solution is lower than HTK solution but was sufficient to maintain the pH during 24 h of hypothermic perfusion. The BGP–HMP solution could be saturated with medicinal air and deliver the adequate O 2 to maintain the low respiration rate of rat livers at 5 or 10 °C.

Biseko® colloidal solution diminishes the vasoreactivity of human isolated radial arteries☆

Radial arteries are increasingly used as grafts in coronary artery bypass surgery. The surgical preparation and intraoperative management of this conduit artery may affect its early and long-term patencies. We investigated the effects of the colloidal Biseko and 5% albumin solutions as well as the crystalloid physiological saline (0.9% NaCl) and Bretschneider solutions on the contractile and relaxing capacities of isolated human radial artery grafts. Radial artery segments were harvested using the technique with an ultrasonic scalpel, and 2.5-3mm rings were obtained from the proximal part of the artery. Arterial rings were stored in Biseko or 5% albumin solutions and in 0.9% NaCl or Bretschneider solutions for 45 min. Isometric tensions of radial arteries obtained from 26 patients were measured in isolated organ baths. Contractions were induced by 0.31 micromolL(-1) 5-hydroxytryptamine and 10 micromol L(-1) noradrenaline. Endothelium-dependent relaxations were induced by 10 micromol L(-1) acetylcholine and 1 micromol L(-1) bradykinin as well as the endothelium-independent relaxations by 10 micromol L(-1) glyceryl trinitrate and 100 micromol/l papaverine. Contractions of radial arteries induced by 5-hydroxytryptamine were significantly lower following storage in Biseko solution (12.6+/-4.4 mN) than in 5% albumin (37.9+/-13.0 mN, p=0.03) or in 0.9% NaCl solution (35.9+/-11.9 mN, p=0.04). Noradrenaline-induced contractions of the arteries were also diminished in Biseko solution compared to those stored in 5% albumin (32.9+/-6.2 mN vs 49.2+/-6.4 mN, p=0.01). No significant differences in relaxations were obtained between the two crystalloid and the two colloidal solutions using endothelium-dependent and independent vasorelaxants. Our results suggest that storage of radial artery in Biseko colloidal solution before coronary artery bypass grafting decreases the sensitivity of the graft to vasoconstriction, thereby decreasing the risk of intra/perioperative graft failure.

Albumin Augmentation Improves Condition of Guinea Pig Hearts After 4 hr of Cold Ischemia

Major causes of death after heart transplantation are right ventricular pump failure and, chronically, cardiac allograft vasculopathy. Traditional preservation techniques focus on immediate cardioplegia, without particularly considering vascular demands. Recently, the endothelial surface layer, composed of the endothelial glycocalyx and plasma proteins, was discovered to play a major role in vascular barrier function, edema formation, and leukocyte-to-endothelial interaction. The impact of augmenting a traditional preservation solution with plasma colloid albumin was therefore investigated. Guinea pig hearts underwent cold ischemic storage for 4 hr using Bretschneider's solution (histidine-tryptophan-ketoglutarate [HTK]) without and with augmentation with 1 g% human albumin. After reperfusion, intracoronary adhesion of polymorphonuclear granulocytes, edema formation, left and right heart performance of pressure-to-volume work, and glycocalyx shedding were assessed. Intracoronary retention of leukocytes was doubled in the traditional group (36.4+/-6.6%), whereas it remained at basal values after albumin preservation (23.5+/-2.4%; P<0.05). Addition of albumin to HTK significantly decreased edema formation (wet to dry weight ratio 6.9+/-0.1 vs. 7.2+/-0.2; P<0.05). Although left heart performance was comparable, right heart cardiac output was doubled in hearts having received HTK containing albumin versus HTK alone (94+/-14 vs. 50+/-11 mL/min/g; P<0.05). Glycocalyx shedding was significantly reduced when the hearts were stored under albumin protection. Augmenting HTK with human albumin improves endothelial integrity and heart performance after 4 hr cold ischemia, because of a marked protection of the endothelial glycocalyx. For the prevention of acute and chronic graft failure, the glycocalyx might represent a new target.

Evaluation of Custodiol N for lung preservation in a porcine lung transplantation model

Objectives: Lung preservation is still a major issue in lung transplantation. Improvements are urgently needed with regard to the high occurrence of I/R injury with subsequent graft failure. The aim of the current study was to evaluate a modified Bretschneider solution (Custodiol N) for lung preservation.

Apoptosis Inhibition for improved Left Ventricular Function after prolonged Cold Cardioplegic Arrest

Cardioplegic arrest (CA) is associated with myocardial apoptosis induction. Data suggest that apoptosis inhibition during regional myocardial ischemia reduces infarct size and improves regional contractility. 28 adult rats were anesthetized and mechanically ventilated. Hearts were arrested by administration of ice-cold crystalloid cardioplegia (Bretschneider solution, Custodiol®, 10 ml/kg) with and without supplementation of a cell permeable non-selective caspase-inhibitor (z-VAD-fmk, 10 mM, n=7 each). Hearts were stored for 4h and 18h (n = 14 each) in cardioplegia (4°C). Subsequently, hearts were reperfused on a Langendorff-System. A balloon connected to a pressure transducer was inserted in the LV and inflated to a diastolic pressure of 10 mmHg. After 4h of cold CA, hearts with apoptosis-inhibition had higher LV pressures throughout the 60 min reperfusion as compared to those without apoptosis-inhibition. Both positive and negative dp/dtmax were significantly higher with apoptosis inhibition. After 18h cold CA, hearts with apoptosis-inhibition had higher left ventricular pressures throughout 90 min reperfusion as compared to those without apoptosis-inhibition. Dp/dtmax was significantly higher with apoptosis inhibition at 70, 80, and 90 min reperfusion. In hearts with apoptosis-inhibition heart rate recovered markedly faster. Apoptosis-inhibition preserves LV function after prolonged cold CA.

Comparative Evaluation of Two Perfusion Solutions for Liver Preservation and Transplantation

The University of Wisconsin solution (UW) and the Bretschneider solution (HTK) were used in 39 adult cadaveric donors: 22 perfused with UW (group 1) and 17 with HTK (group II). Donors were flushed through the aorta (UW, 5 to 6 L; HTK, 8 to 10 L) and through the portal vein (UW or HTK, 1 L). Grafts perfused with HTK showed lower levels of SGOT at postoperative day 7 than those transplanted with UW (38 ± 19 vs 58 ± 31, P < .05). No difference was observed in other functional and outcome parameters. No cases of primary dysfunction were observed. Six-month graft survival was 85.7% in HTK group and 80.9% in UW group ( P = NS). Six unrelated deaths were observed. Five biliary complications were observed in five patients: three in the UW group and two in the HTK group. In conclusion, data fail to show major differences between the two solutions used.

Effect of Four Crystalloid Cardioplegias on Immature Rabbit Hearts During Global Ischaemia

Controversy surrounds the reported beneficial effects of crystalloid cardioplegic solutions in the immature myocardium. In the present study, we investigated the efficacy of four clinical cardioplegic solutions in the immature myocardium to determine if cardioplegic protection could be demonstrated and, if yes, the relative efficacy of the four solutions. Isolated, working hearts (n=6 per group) from neonatal rabbits (age, 7-14 days) were perfused aerobically (37 C) for 15 minutes in the Langendorff mode and 30 minutes in the working mode before a 2-minute infusion of one of four cardioplegic solutions: the modified St. Thomas' Hospital no. 1 cardioplegic solution, Tyers solution, Bretschneider solution or Roe solution. Hearts were then rendered globally ischaemic for 120 minutes at 14C before reperfusion for 15 minutes in the Langendorff mode and 30 minutes in the working mode. The post-ischaemic recovery of cardiac function and leakage of myocardial enzymes (GOT, CK, CK-MB, LDH, LDH1) were compared with results in non-cardioplegic control hearts. Good protection was observed with modified St. Thomas' Hospital and Tyers solutions: postischaemic recovery of cardiac output was increased from 80.43+/-3.62% in the non-cardioplegic group to 85.19+/-3.12% and 70.66+/-3.48% in the St. Thomas' Hospital and Tyers groups (p<0.05), respectively. In contrast, no obvious protection was observed with either the Bretschneider or Roe solutions: cardiac output recovered to 45.08+/-3.16% and 30.06+/-2.59%, respectively. Post-ischaemic CK leakage was 19.83+/-2.14 IU/mL and 21.17+/-2.32 IU/mL in the St. Thomas' Hospital and Tyers groups (p>0.05). In the Bretschneider group, CK leakage increased to 30.00+/-3.16 IU/mL (p<0.01 vs. non-cardioplegic control hearts), and in the Roe group, CK leakage was 31.00+/-5.10 IU/mL (p<0.05 vs. cardioplegic-free hearts). Post-ischaemic ATP was 1.98+/-0.54 micromol/g*dry weight and 1.35+/-0.39 micromol/g*dry weight in the St. Thomas' Hospital and Tyers groups (p<0.01 vs. non-cardioplegic control group), respectively. In the Bretschneider group, ATP decreased to 0.91+/-0.16 micromol/g*dry weight (p<0.05 vs. non-cardioplegic control hearts), and in the Roe group to 0.88+/-0.10 micromol/g*dry weight (p<0.01 vs.cardioplegic-free hearts). In conclusion, cardioplegic protection can be achieved in the immature rabbit myocardium with both St. Thomas' Hospital and Tyers solutions, but acalcaemic solutions such as Bretschneider and Roe solutions increased damage. The reported lack of cardioplegic efficacy in the immature myocardium may, therefore, reflect the choice of cardioplegic solution rather than a greater vulnerability to injury in the neonatal heart.

Apoptosis Inhibition Improves Left Ventricular Function After Prolonged Cold Cardioplegic Arrest

Cardioplegic arrest (CA) is associated with myocardial apoptosis induction. Apoptosis inhibition during regional myocardial ischemia reduces infarct size and improves regional contractility. We investigated whether inhibition of the apoptosis-signal-pathway improves left ventricular (LV) function following prolonged cold CA. Ten adult rat hearts were arrested using ice-cold cardioplegia (Bretschneider solution, Custodiol®, 10 ml/kg) with and without supplementation of a non-selective caspase-inhibitor (z-VAD-fmk, 10 mM, n=5 each). Hearts were stored for 4h in cardioplegia (4°C). Subsequently, hearts were reperfused with Krebs-Henseleit-Solution (37°C) on a Langendorff-System. Five additional rats without ischemic storage served as controls. After 4h cold CA, hearts with apoptosis-inhibition had higher left ventricular pressure throughout the 60min reperfusion as compared to those without apoptosis-inhibition. Apoptosis-inhibition even resulted in significantly higher LV pressures as compared to controls at 30, 40 and 50 min reperfusion (58.6±10, 59.2±8.1 and 58.6±8.6 mmHg vs. 42±8.7, 42.8±10.6 and 42.2±8.6 mmHg, respectively; p<0.013). Recovery time on reperfusion defined as duration until heart rate and LV pressure reached steady state was significantly shorter with apoptosis inhibition as compared to non-inhibition (13.4±11.6 vs 29.6±8.2 min, p=0.017). Apoptosis-inhibition improves LV-function after prolonged cold CA and represents a new promising strategy in myocardial protection for cardiac allografts.

Effect of hypothermia and HTK on the microcirculation in the rat cremaster muscle after ischaemia

Hypothermia is an important preservation method for tissues and solid organs. The aim of the present study was to assess in rat cremaster muscle the effect of hypothermia, without or with pre-ischaemic HTK (histidine-tryptophan-ketoglutarate-Bretschneider solution) perfusion, on microvascular consequences of 4 or 6 h ischaemia and 2 h of reperfusion. Intravital microscopy was applied to examine capillary perfusion and leucocyte-endothelium interactions. The cremaster muscle was subjected to 4 or 6 h of cold (4 degrees C) or warm (33-34 degrees C) ischaemia and 2 h of reperfusion. Measurements were performed at baseline, prior to HTK perfusion and ischaemia, and at 0, 1 and 2 h after blood flow restoration. Hypothermia completely prevented the 50% reduction in capillary perfusion that was observed previously at start of reperfusion after 4 h warm ischaemia. After 6 h of warm ischaemia, perfusion resumed in only 45% of capillaries and remained at this low level during reperfusion. In contrast, only a slight decrease (< 10%) in capillary perfusion was observed after 6 h of cold ischaemia. Pre-ischaemic HTK perfusion had no beneficial effect on tissue perfusion. Both hypothermia and HTK attenuated the significant increase in venular leucocyte-vessel wall interactions, which was observed after 4 h of warm ischaemia in a previous study. Combined application of both interventions had no additional effects. After 6 h of warm ischaemia, no increase in leucocyte-vessel wall interactions was observed, possibly due to venular flow reduction. In conclusion, hypothermia preserves capillary perfusion and prevents an increase in leucocyte-vessel wall interactions during reperfusion after muscle tissue ischaemia. Preischaemic perfusion of the vasculature with HTK does not improve the effects of cold storage on tissue perfusion, but attenuates the inflammatory response independently of temperature effect.

EVALUATION OF INITIAL RENAL FUNCTION AFTER KIDNEY TRANSPLANT IN ISOGENEIC WISTAR RATS. COMPARATIVE ANALYSIS OF WISCONSIN AND HTK MEDIA AS PERFUSION SOLUTION OF DONOR KIDNEY.

P1212 Aims: In order to compare Wisconsin and Bretschneider solutions in immediate evolution of renal function, oxidative stress and morphology of kidney transplant in syngeneic Wistar rats, the following experiment was performed. Methods: Eight syngeneic rats were submitted to kidney transplants by conventional technique. Before transplantation, 4 kidneys were perfused with Wisconsin solution and the remaining 4 with HTK solution. The cold ischemia time was 4 hours. By day 3 post transplant (p.t.), native kidneys were removed. On day 6 p.t. rats were sacrificed and blood samples were obtained as well as the grafts for histological examination. Creatinine was measured by colorimetric assay in blood samples obtained prior to transplant, at day 3 p.t. and at the moment of sacrifice. Nitric oxide was measured as nitrite/nitrate using a commercial kit (R&D Systems Nitric Oxide Assay) prior to transplant and at day 6 p.t. Results: All transplant procedures were successful, surviving animals until the end of the experiment, with minor morbidity. Macroscopically, all grafted kidneys show a normal aspect and histologically no lesions were found. Pre-transplant creatinine value (expressed as a mean) was around 0.73 mg/dl for both groups. By day 3 p.t. creatinine levels were 1.14 mg/dl and 0.91 mg/dl for Wisconsin and HTK treated animals, respectively. By day 6 p.t. creatinine levels increased to 1.29 and 1.03 mg/dl, respectively (difference: N.S.). Pre-transplant NO value (expressed as NO2/NO3 and as a mean of 8 rats) was 126.5 Umol/l. By day 6, average ON level dropped to 67.6 Umol/l in HTK treated animals and to 33.0 Umol/l in Wisconsin treated group. Conclusions: As expected neither rejection signs nor lesions were found in this group of syngeneic animals. Creatinine slowly increased in time in HTK treated animals, Increase in creatinine levels in Wisconsin treated animals was higher but statistically not significant. Nevertheless, creatinine levels were not so high in both groups indicating a good renal function after transplant. Both solutions reduced ON levels by the end of the experiment. More studies will be necessary in order to better evaluate differences between both solutions.

Coronary oxygen persufflation for heart preservation in pigs: analyses of endothelium and myocytes.

Coronary oxygen persufflation (COP) has been shown to prolong heart preservation time up to 14 hr in a mature pig model, with excellent recovery after orthotopic transplantation. The aim of the present study was to assess the structural, metabolic, and functional myocardial and endothelial integrity after COP in mature pig hearts. Cardioplegic arrest was induced by original crystalloid Bretschneider solution (HTK 3h, n=6), modified Bretschneider solution (mHTK+COP, n=6), or University of Wisconsin solution (UW+COP, n=6). Hearts were stored for 3 (HTK 3h) or 14 hr (mHTK+COP, UW+COP) at 0 degrees to 1 degrees C. In addition, COP hearts were persufflated. After heterotopic transplantation and reperfusion for 7 days, hearts were analyzed by light microscopy or electron microscopy for structural injuries. Endothelial function, cardiac enzymes, metabolic parameters, and myocardial water content (MWC) were determined. Six recipient hearts served as controls. Quantitative light microscopic analyses and semiquantitative electron microscopic analyses showed an equal amount of damage in all groups including HTK 3h hearts. No rejection was observed. Substance P induced an equal dilatation in all hearts. Serum levels of cardiac enzymes were similar in all groups, but energy-enriched phosphates were significantly reduced, and MWC was augmented in the HTK 3h hearts and in the UW+COP hearts, in contrast to the mHTK+COP transplants. The lack of structural defects related to the COP technique, similar endothelial function, and an even better metabolic state of the mHTK+COP hearts versus HTK 3h hearts demonstrate the efficacy of the COP technique for prolongation of myocardial preservation time up to 14 hr.

Viability of the myocardium after twenty-four-hour heart conservation--a preliminary study.

Orthotopic heart transplantation following ischemic times beyond four hours is associated with increased risk of early graft failure. The use of modern myocardial preservation strategies could enable safe transplantation after long-term conservation. In this study, we tested a new myocardial protection regime in an experimental model of 24 h storage. Orthotopic heart transplantations (n=15) were performed in a pig model. Donor hearts were flushed with Bretschneider solution, excised, and stored for 24 hours at 4 degrees C. During implantation, controlled reperfusions with substrate-enriched leukocyte-depleted blood cardioplegia were performed after each anastomosis. Blood cardioplegia contained 1 mmol/l of the Na(+)-H(+)-exchange inhibitor HOE 642 and 100 mg/l of adenosine. Controlled reperfusion was continued with leukocyte-depleted blood for 20 min. A microaxial pump was inserted after heart transplantation and circulatory assistance was maintained for five hours to prevent right heart failure. No initial graft failure could be observed. Thirteen hearts could be weaned from extracorporeal circulation. Due to bleeding problems, kidney and lung failure only five hearts could be included in the final analysis. Hemodynamics of these hearts remained stable with epinephrine at 0.1 micro g/kg/min. Myocardial oxygen consumption 20 min after start of reperfusion (5.3+/-2.0 ml/100 g/min) did not differ significantly versus baseline (6.8+/-2.0 ml/100 g/min). Oxygen extraction six hours after heart transplantation was also well preserved compared to baseline (58.0+/-10.2 versus 49.2+/-8.8 %). Histological examination six hours after transplantation using luxol fast blue staining revealed that only 1.0 % of the myocytes were irreversibly damaged. The data indicate full viability of the myocardium after 24 h conservation. The preservation technique described could contribute to the extension of conservation times in heart transplantation and enable transplantation of marginal donor hearts.

Improved results after heart-lung transplantation: a 17-year experience

In selected patients with severe end-stage combined cardiopulmonary diseases, heart-lung transplantation (HLTx) remains the only therapeutical option for improving survival and quality of life. Since 1983, 51 HLTx were done at our institution. Mean patient age was 27+/-12 years with a mean donor age of 25+/-11 years. Indications for HLTx were primary pulmonary hypertension (PPH) in 49% of patients, congenital heart disease in 39%, cystic fibrosis in 6%, and repeat-HLTx in 6%. Eleven patients were younger than 14 years. Among these pediatric patients, the indications were PPH in 55% of patients, pulmonary atresia with severe pulmonary artery hypoplasia in 27%, and cystic fibrosis and cardiomyopathy with fixed pulmonary hypertension in 9% of patients each. Two patients had additional liver transplantation because of chronic aggressive virus hepatitis. For organ preservation, Euro-Collins solution (lung perfusion) and cardioplegic solution according to Bretschneider (heart perfusion) were used until 1994. The University of Wisconsin solution replaced Bretschneider's solution in 1994. Since 1996, Perfadex, a low-potassium dextran-based preservation solution, replaced Euro-Collins. All transplantations were done through a median sternotomy until 1994. Thereafter, a transverse thoracotomy was used in patients with suspected adhesions. Until 1995, cyclosporine A, azathioprine, and prednisolone were used for immunosuppression. Since then, tacrolimus replaced cyclosporine A. From 1983 until 1993, perioperative mortality was 35% (6/19). From 1994 on perioperative mortality decreased to 12.5% (4/32). Early mortality was caused by graft failure (n=5), severe bleeding (n=2), multi-organ failure (n=2), and acute rejection (n=1). Cumulative survival rates were 81% after 30 days, 63% after 1 year, and 54% after 5 years, respectively. Since 1994, cumulative survival rates were markedly improved to 87% after 30 days, 81% after 6 months, and 78% after 1 year. There was no death during the first postoperative year among the 11 pediatric patients. Late death was mainly caused by obliterative bronchiolitis (OB; 76%); two patients died because of multi-organ failure or septic complications, respectively, and one patient died within the first postoperative year because of aspergillosis. Changes in organ preservation management, surgical techniques, and immunosuppressive therapy significantly improved the short- and mid-term results after HLTx. Long-term results can only be improved in cases of successful prevention and treatment of OB.