Proteins released from liver after ischaemia induced an elevation of heart resistance against ischaemia-reperfusion injury: 2. Beneficial effect of liver ischaemia in situ

We have shown earlier that proteins released from the heart during preconditioning may protect non-preconditioned heart during sustained ischaemia, similarly as preconditioning itself. In other our experiments we have documented that also proteins released from isolated rat liver during reperfusion after global ischaemia performed a protective effect on isolated rat heart against ischaemia-reperfusion injury. In the current study we examined the effect of liver ischaemia in situ on resistance of rat heart to ischaemia and reperfusion injury. Wistar rats (male) were subjected to liver ischaemia maintained by occlusion of portal vein and hepatic artery for 20 min, followed with 30-min reperfusion after reopening of both vessels. Then the hearts were isolated and perfused according to Langendorf. Hearts, after initial stabilisation (15 min), were subjected to 20-min ischaemia and 30-min reperfusion. During reperfusion, the haemodynamic parameters of hearts were measured. The protein pattern of high soluble fraction (HS fraction) isolated from rat blood by precipitation with ammonium sulphate was detected by SDS-PAGE. Our results showed improved parameters of pressure and contractility in the group after liver ischaemia (ischaemic group), presented by decreased diastolic pressure and increased LVDP((S-D)) in comparison with levels of these parameters in the control group. We also observed improved heart contraction-relaxation cycles parameters (dP/dt)(max) and (dP/dt)(min) in ischaemic group as compared with the control group. On the other hand, there were no significant differences in heart rate and coronary flow between both experimental groups. SDS-PAGE showed changed protein pattern in HS fraction, particularly the levels of several low molecular weight proteins increased. We conclude that liver ischaemia induced a higher resistance of heart against ischaemia-reperfusion injury. We propose that release of some cardioprotective proteins present in HS fraction can also contribute to this cardioprotection.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Donors of nitric oxide mimic effects of ischaemic preconditioning on reperfusion induced arrhythmias in isolated rat heart

NO has been implicated in the mechanism of ischaemic preconditioning. To verify this hypothesis further we have attempted to reproduce effects of ischaemic preconditioning by nitric oxide donors administration prior to the ischaemia. The effect of glyceryl trinitrate (GTN) and 3-morpholino-sydnonimine-hydrochloride (SIN- 1), NO donors, on reperfusion induced ventricular tachycardia (VT) and ventricular fibrillation (VF) in Langendorff perfused rat hearts subjected to 10 min regional ischaemia followed by 10 min reperfusion were examined. Results: GTN, 500 M and SIN-1, 10 M, administered for 5 min and washed for another 5 min prior to ischaemia (to mimic ischaemic preconditioning), almost completely abolished reperfusion induced VF. GTN and SIN-1, administered at the time of reperfusion, increased the incidence of sustained VF and the duration of VT and VF. When given 5 min before the ischaemia and throughout the ischaemia and the reperfusion, SIN-1 abolished VF. Adenosine, 10 M, applied according to the above three protocols, did not affect reperfusion arrhythmias, although adenosine induced changes in coronary flow and post-ischaemic reflow were similar to those produced by the NO donors. In conclusions: (1) NO is able to mimic the effect of ischaemic preconditioning on reperfusion arrhythmias in rat heart, supporting the view that NO may be one of the endogenous substances triggering ischaemic preconditioning; (2) In crystalloid-perfused heart, NO may be deleterious when its administration is restricted to the reperfusion period.     

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.     

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Electrophysiological studies with repeated episodes of ischaemia on isolated rat heart

In order to know the beneficial effect of preconditioning electrocardiography recording were used as tool to assess myocardial malfunction and for this perfusion apparatus was setup. Electrophysiological changes for each heart were recorded during perfusion at 1, 2, 3, 5, 10, 20, 30 and 60 min of global ischaemia and also during the equal period of reperfusion. Recordings dembnstrate that the normal rate was about 240 beats/min with an "R" amplitude of 4mV. During the first ischaemic episode of 1min the rate was 180 +/- 15 beats/min (counted as per 'R' wave deflection), at 2 mins it was 60 +/- 6 beats/min, at 3 min the rate was 40 +/- 2 beats/min, at 5 mins of ischaemia it was 90 +/- 6 beats/min, at 10 min 20 +/- 2 beats/min, at 20 min the rate was 60 +/- 4 beats/min, and at 30 mins there were nil beats/min. The recovery during all the periods of reperfusion was restored to between 120 and 180 beats/min in all episodes. Further after a 60 min of ischaemia the heart stopped to elicit any mechanical response. It is concluded that short term ischaemia can induce a resilient effect on the beating of the heart after a few episodes as seen subsequent to 1 and 2 min of ischaemia. Further, preconditioning was beneficial up to 30 min, beyond which the heart showed signs of fatigue and irreversible injury.     

Protective effects of dexrazoxane against acute ischaemia/reperfusion injury of rat hearts

Dexrazoxane (DEX), an inhibitor of topoisomerase II and intracellular iron chelator, is believed to reduce the formation of reactive oxygen species (ROS) and protects the heart from the toxicity of anthracycline antineoplastics. As ROS also play a role in the pathogenesis of cardiac ischaemia/reperfusion (I/R) injury, the aim was to find out whether DEX can improve cardiac ischaemic tolerance. DEX in a dose of 50, 150, or 450?mg·(kg body mass)(-1) was administered intravenously to rats 60?min before ischaemia. Myocardial infarct size and ventricular arrhythmias were assessed in anaesthetized open-chest animals subjected to 20?min coronary artery occlusion and 3?h reperfusion. Arrhythmias induced by I/R were also assessed in isolated perfused hearts. Only the highest dose of DEX significantly reduced infarct size from 53.9%?± 4.7% of the area at risk in controls to 37.5%?± 4.3% without affecting the myocardial markers of oxidative stress. On the other hand, the significant protective effect against reperfusion arrhythmias occurred only in perfused hearts with the dose of DEX of 150?mg·kg(-1), which also tended to limit the incidence of ischaemic arrhythmias. It is concluded that DEX in a narrow dose range can suppress arrhythmias in isolated hearts subjected to I/R, while a higher dose is needed to limit myocardial infarct size in open-chest rats.     

Adenine nucleotide synthesis from inosine during normoxia and after ischaemia in the isolated perfused rat heart

[14C]inosine in a range of concentrations of 20 microM to 1 mM was administered to the isolated perfused rat heart for 30 min. The incorporation of the nucleoside into myocardial adenine nucleotides increased for extracellular concentrations of the precursor up to 50 microM, reaching a plateau at 60 nmol . g-1 X 30 min-1 with concentrations ranging between 50 and 200 microM. The supply of 500 microM and 1 mM of inosine induced a further increase in cardiac adenine nucleotide synthesis to about 200 nmol . g-1 X 30 min-1. When supplied during low flow ischaemia (0.5 mL . min-1, 30 min.), 1 mM of inosine protected the heart against ATP degradation, while 100 microM of inosine was inefficacious. In the presence of 1 mM of inosine on reperfusion the adenine nucleotide content of the heart was similar to that observed in the absence of the nucleoside. The incorporation of [14C]inosine into adenine nucleotides was, in this last condition, below the value measured before ischaemia. Inosine administration was effective in protecting the heart against ischaemic breakdown of glycogen and favoured postischaemic restoration of glycogen stores.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

The influence of lactate,pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p<0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p<0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p<0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p<0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups. Thus, the endogenous defense mechanism against oxygen-derived free radicals is compromised at the onset of reperfusion when glucose as sole substrate is present, while addition of lactate or pyruvate prevents reduction of the endogenous capacity to scavenge oxygen-derived free radicals. The equivocal relationship between endogenous scavenging enzyme activity and haemodynamic recovery indicates that involvement of the endogenous antioxidants, if any, in functional recovery of the post-ischaemic heart is complex. Pyruvate may exert protective effects on mechanical function after mild ischaemia by functioning as exogenous scavenger in itself, as pyruvate is able to react with hydrogen peroxide.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Acid hydrolase activity and cyclic nucleotide contents in the rat heart during myocardial ischemia and postischemic reperfusion

The acid phosphatase and cathepsin D activities and cAMP and cGMP levels in isolated perfused rat heart were investigated during various periods of ischaemic myocardial injury and postischaemic reperfusion. The effect of phosphodiesterase inhibitor--caffeine was also studied. Free acid hydrolases activities and cyclic nucleotide content were increased under 40 and 60 min ischemia and 20 min postischaemic reperfusion. Addition of 50 microM caffeine to perfusion solution after 30 min of ischaemia resulted in increase of cAMP level, cAMP/cGMP ratio, lysosomal bound activities of acid hydrolase and decrease of free acid hydrolase activities. The obtained results suggested that defect in cAMP synthesis might be present in lysosomal membranes labilization in cardiomyocytes injured during ischaemic conditions. Addition of such agents, as caffeine, which increased heart cAMP level, may be effective in lysosomal membranes stabilization under reversible heart ischaemia and reperfusion.     

丙泊酚预处理对大鼠离体心肌浅低温缺血／再灌注损伤后线粒体细胞色素C释放的影响

目的：探讨丙泊酚预处理对大鼠离体心肌浅低温缺血／再灌注（I／R）损伤后心肌细胞凋亡及线粒体细胞色素C释放的影响。方法：应用Langendorff离体心脏灌注模型,取50只SD大鼠随机分为5组：对照组（C组）,二甲基亚砜（DMSO）预处理组（D组）,25、50、100μmol·L^-1丙泊酚预处理纽（P1、P2、P3组）。各组均浅低温缺血55min,再常温灌注60min。D组、P1、P2、P3组在缺血前分别以含DMSO、相应浓度丙泊酚的K-H液灌注10min,再冲洗5min,重复2次。记录平衡灌注末、缺血前即刻、再灌注30、60min时的心功能指标。再灌注60min时测定凋亡细胞,提取心肌线粒体,测定线粒体和胞浆的细胞色素C水平。结果：与C组相比,P3组再灌注30min、60min时左室舒张末压（LVEDP）降低、左室发展压（LVDP）升高（P〈0．05或P〈0．01）;P2、P3组再灌注末心肌细胞凋亡率降低（P〈0．05或P〈0．01）,线粒体细胞色素c释放减少,胞浆细胞色素C的量明显降低（P〈0．05或P〈0．01）。结论：丙泊酚预处理能够通过抑制心肌线粒体细胞色素C释放到胞浆,降低浅低温I／R损伤心肌细胞凋亡率,减轻心肌桶伤．     

Thyroid hormone and cardioprotection: Study of p38 MAPK and JNKs during ischaemia and at reperfusion in isolated rat heart

It has been recently shown that long-term thyroxine administration increases the tolerance of the heart to ischaemia. The present study investigated whether thyroxine induced cardioprotection involves alterations in the pattern of p38 mitogen activated protein kinase (p38MAPK) and c-Jun NH₂-terminal kinases (JNKs) activation during ischaemia-reperfusion. L-thyroxine (T4) was administered in Wistar rats (25 g/100 g/day, subcutaneously) for 2 weeks (THYR), while normal animals served as controls (NORM). NORM and THYR isolated rat hearts were perfused in Langendorff mode and subjected to 10 or 20 min of zero-flow global ischaemia only and also to 20 min of ischaemia followed by 10, 20 or 45 min of reperfusion. Postischaemic recovery of left ventricular developed pressure at 45 min of reperfusion was expressed as % of the initial value. Activation of p38 MAPK and JNKs was assessed at the different times of the experimental setting by standard Western blotting techniques using a dual phospho p38MAPK and phospho JNKs (p46/p54) antibodies. Activation of p38 MAPK was significantly attenuated during ischaemia and reperfusion in thyroxine treated hearts compared to normal hearts. JNKs were found to be activated only during the reperfusion period. The levels of phospho JNKs were found to be lower in thyroxine treated hearts as compared to untreated hearts, though not at a statistically significant level. Postischaemic functional recovery was higher in THYR as compared to NORM, p < 0.05. In summary, in hearts pretreated with thyroxine, p38 MAPK was attenuated during ischaemia and at reperfusion and this was associated with improved postischaemic recovery of function.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

The isolated perfused rat liver: standardization of a time-honoured model

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms.A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function).The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs-Henseleit buffer, without albumin. The usual volume is 150-300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37 degrees C. Standardized markers should be used to allow comparison with other experiments.     

Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation

A number of works show that the mitogen-activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed-lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.     

Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells

The present study is designed to investigate the effect of myocardial preconditioning with oxidative stress induced by pyrogallol or H₂O₂, on ischaemia-reperfusion induced myocardial injuiry. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of preconditioning induced by pyrogallol or hydrogen peroxide (H₂O₂) or ischaemia markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. Administration of polymyxin B, a protein kinase C (PKC) inhibitor, during pyrogallol, H₂O₂ or ischaemic preconditioning markedly attenuated the cardioprotective effect of preconditioning produced with oxidative stress or ischaemia. These results suggest that preconditioning with oxidative stress may provide cardioprotection similar to ischaemic preconditioning, against ischaemia-reperfusion injury and this cardioprotective effect may be mediated through activation of PKC.     

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.