Histamine release and its effects in ischaemia-reperfusion injury of the isolated rat heart

Histamine is released from the heart during ischaemia-reperfusion injury. As histamine has cardiac effects, we investigated the role of histamine in ischaemia-reperfusion injury of isolated rat hearts. A Langendorff-model with 30 min global (37 ^oC) ischaemia followed by 60 min reperfusion was employed. The effects of ischaemia alone (n= 10, group 1.1+n = 10, group 2.1, 2 different series), and ischaemia with H₁- and H₂-receptor blockade with cimetidine (10 μM, n= 10), chlorpheniramine (10 μm, n= 8), terfenadine (10 μM, n= 8), and promethazin (10 μM, n= 9), or both cimetidine and chlorpheniramine (n = 8), were studied. Histamine was measured in the coronary effluent and cardiac tissue of group 1.1. Release of histamine increased from 6.5 ± 1 pmol min^-1 before ischaemia to 19 ± 3 pmol min^-1 at the start of reperfusion. Ischaemia decreased left ventricular developed pressure to 18 ± 11 % (1.1) and 50 ± 11 % (2.1) of initial value (mean ± SEM) at the start of reperfusion. Left ventricular end-diastolic pressure increased from 0 to 79 ± 8 mmHg (1.1) and 39 ± 9 (2.1) mmHg, while left ventricular systolic pressure was unchanged (101 ± 12% in 1.1 and 101 ± 10% in 2.1). Severe arrhythmias were induced in 90 (1.1) and 30 (2.1)% of the hearts, while coronary flow decreased during reperfusion. H₂-blockade did not modify the changes in left ventricular pressures, coronary flow, or heart rate induced by ischaemia. Three different Hj-blockers increased left ventricular systolic pressure, inhibited the decrease of developed pressure, attenuated the increase of end-diastolic pressure, and totally inhibited reperfusion arrhythmias. The effect of both blockers together was similar to that of H₁-blockers alone. Coronary flow was increased during reperfusion in two of the groups with Hj-blocker compared with ischaemic controls. Increased release of histamine from ischaemic-reperfused rat hearts concurred with depression of left ventricular function and arrhythmias during early reperfusion. Cardiac dysfunction during reperfusion was attenuated by three different Hj-receptor blockers.     

Sulfonylureas enhance GABAA synaptic potentials in rat midbrain dopamine neurones

Long-lasting myocardial ischaemia reduces the density of sarcolemmal L-type calcium channels (LCC). Ischaemic preconditioning protects the myocardium against development of infarction. The aim of this study was to investigate if ischaemia-induced loss in LCC is affected by ischaemic preconditioning. Specific (+)-[³H]isradipine binding to LCC was compared in membranes and homogenates from control and ischaemic regions of non-preconditioned and ischaemically preconditioned hearts [two 10 min left anterior descending coronary artery (LAD) occlusions, each followed by 30 min reperfusion]. Biopsies were sampled after 60 min mid LAD occlusion from ischaemic and control (supplied by circumflex artery) regions. Sixty min ischaemia reduced binding density of specific (+)-[³H]isradipine in membranes by 23±11% (n=7, P<0.05) in the non-preconditioned group and by 20±8% (n=6, P<0.05) in the preconditioned group. Binding density in homogenates was reduced by 36±5% (n=5, P<0.05) in the non-preconditioned group and by 21±5% (n=5, P<0.05) in the preconditioned group. The reductions in the two groups and reductions in membranes and homogenates were not statistically different. The dissociation constant of binding was similar in the groups. In conclusion, 60 min of ischaemia reduced the binding density of (+)-[³H]isradipine in membranes and homogenates by 20–36%. The reduction in density of binding sites was not caused by redistribution of sarcolemmal LCC to an intracellular compartment. Ischaemic preconditioning did not affect the decline in binding density as hypothesized.     

High afterload during 10 min of regional ischaemia affects diastolic creep but not systolic function in reperfused (stunned) myocardium

The effect of afterload during regional ischaemia on myocardial stunning was studied in 15 pentobarbital anaesthetized cats. 10 min occlusion of the left anterior descending artery (LAD) was followed by 60 min of reperfusion. Afterload was decreased by intravenous infusion of nitroglycerine 3–8 μg kg^-1 min^-1 in group I (n=8); left ventricular peak systolic pressure (LVSP) 84±4 mmHg (mean±SEM) during coronary artery occlusion. In group II (n=7) LVSP was increased to 188±10 mmHg by inflating an intraaortic balloon during coronary artery occlusion. Regional function in the LAD perfused region was evaluated by cross-oriented sonomicrometry. Myocardial tissue blood flow was evaluated by radio-labelled microspheres. Afterload alterations did not affect regional systolic shortening (10.8±2.0% vs. 11.0±1.5% in group I and II, respectively, after 60 min of reperfusion). However, increased end-diastolic dimensions (diastolic creep) in both the circumferential and longitudinal segments were markedly more pronounced in the high afterload group (group II). Also important, the markedly increased myocardial tissue blood flow during reperfusion in group II as compared with group I (2.30±0.18 vs.  1.34±0.08 mL min^-1 g^-1 and 2.58±0.23 vs. 1.49±0.07 mL min^-1 g^-1 in subepicardial and subendocardial layers in the LAD perfused region) suggests that increased diastolic creep increased metabolic demands. This study indicates that passive stretching of the ischaemic area during coronary artery occlusion is an important mechanism behind diastolic creep.     

The novel non-peptide selective endothelin A receptor antagonist LU 135 252 protects against myocardial ischaemic and reperfusion injury in the pig

The aim of the study was to investigate the efficacy of the novel non-peptide selective endothelin A (ET_A) receptor antagonist LU 135 252 to limit the extent of myocardial ischaemic and reperfusion injury. Administration of LU 135 252 (1 and 5 mg kg^–1 i.v.) to anaesthetised pigs reduced mean arterial pressure (MAP) from 91 ± 4 to 79 ± 3 mmHg (P < 0.05) and 96 ± 3–82 ± 3 mmHg (P < 0.01), respectively. Heart rate, coronary blood flow and coronary vascular resistance were not affected by LU 135 252. The infarct size induced by 45-min ligation of the left anterior descending coronary artery (LAD) followed by 4-h reperfusion in pigs was 81 ± 5% of the area at risk in control animals given vehicle (n = 8). In pigs receiving 1 mg kg^–1 (n = 6) or 5 mg kg^–1 (n = 8) of LU 135 252 i.v. 20 min before ischaemia the infarct size was reduced to 64 ± 3% (P < 0.05) and 35 ± 4% (P < 0.001), respectively, of the area at risk. During the reperfusion period there was a non-significant trend towards a higher coronary blood flow and a lower coronary vascular resistance in the groups given LU 135 252 compared to controls. Myocardial overflow of ET-like immunoreactivity was increased during the reperfusion period but it was not affected by administration of LU 135 252. It is concluded that administration of the selective ET_A receptor antagonist LU 135 252 effectively protects the myocardium from ischaemia/reperfusion injury, indicating that the ET_A receptor subtype is involved in the development of ischaemia/reperfusion injury.     

Peroxisomal fatty acid oxidation capacity is more resistant to ischaemic and reperfusion injury than mitochondrial fatty acid oxidation capacity in feline hearts

We investigated ischaemic and postischaemic mitochondrial and peroxisomal fatty acid oxidation capacity, ATP levels and regional function in 40 anaesthetized open chest cats subjected to 10 or 40 min of regional myocardial ischaemia with or without 3 h of reperfusion (n=10 in each situation). Following 10 min of ischaemia, the mitochondrial fatty acid oxidation capacity measured in tissue extracts from ischaemic tissue (nmol min^-1 mg protein^-1) was reduced in both subepi- and subendocardium, but was normalized in reperfused tissue extracts from both wall layers (0.29±0.03 and 0.30±0.04 vs. 0.57±0.05 and 0.59±0.05, P<0.05). Peroxisomal fatty acid oxidation capacity in tissue extracts was unaffected by ischaemia and reperfusion. ATP levels and regional function measured in the LAD region was partly restored transmurally. After 40 min of LAD occlusion, mitochondrial fatty acid oxidation capacity was reduced, with higher activity in subepi- than in subendocardium (0.27±0.05 vs. 0.19±0.04, P<0.05). Reperfusion did not restore mitochondrial fatty acid oxidation capacity. Peroxisomal fatty acid oxidation capacity was increased in the ischaemic subendocardium compared with levels in non-ischaemic subendocardium (0.53±0.02 vs. 0.45±0.03, P<0.05), with normalization at the end of reperfusion. ATP levels were non-uniformly reduced during ischaemia and not repleted during reperfusion. Regional function recovered in circumferential segments but not in longitudinal segments following 40 min of ischaemia. In conclusion fatty acid oxidation enzymes seem to be more resistant to ischaemia in peroxisomes than in mitochondria. Mitochondrial fatty acid oxidation is fully reversible following shortlasting ischaemia, but remains depressed following prolonged ischaemia and reperfusion.     

Role of l-arginine in preventing myocardial and endothelial injury following ischaemia/reperfusion in the rat isolated heart

The protective effect of l -arginine on ischaemia/reperfusion-induced myocardial injury was investigated in the rat isolated Langendorff perfused heart. Six groups of hearts subjected to 30 min global ischaemia and 30 min reperfusion received either vehicle, d -arginine, l -arginine, the nitric oxide (NO)-donor S-Nitroso-N-Acetyl-d, l -Penicillamine (SNAP), the inhibitor of NO formation N^G-nitro-l -arginine (l -NNA), or l -arginine plus l -NNA. The recoveries of left ventricular double product and coronary flow at the end of reperfusion were significantly higher in the l -arginine group (85±5 and 75±6%, respectively) than in the vehicle group (37±6 and 34±5%, respectively, P<0.05). During both the ischaemic and reperfusion periods, left ventricular end diastolic pressure was lower in the l -arginine group than in the vehicle group. Creatine kinase outflow and the area of no-reflow were smaller in the l -arginine treated hearts (P<0.01). There were no differences between vehicle and d -arginine treated groups. l -NNA did not affect recovery per se but abolished the protective actions of l -arginine. SNAP produced the same protective effects as l -arginine. Acetylcholine-induced endothelium-dependent vasodilation was reduced after ischaemia and reperfusion in the vehicle group but not in the l -arginine group. It is concluded that l -arginine reduces ischaemia/reperfusion-induced myocardial and endothelial injury. The results suggest that the beneficial effects of l -arginine are related to preserved synthesis and release of NO.     

Endogenous nitric oxide attenuates hypoxic vasoconstriction of small pulmonary arteries and veins in anaesthetized cats

Ischaemic preconditioning has cardioprotective effects. Reactive oxygen species may be possible mediators. The present study investigated whether low doses of exogenous hydrogen peroxide could mimic preconditioning in isolated, Langendorff-perfused rat hearts. Hearts were subjected to two episodes of 3 min global ischaemia and 5 min reperfusion (n = 17), or were given 10 (n=15), 20 (n=10), 30 (n=20), 40 (n=18), 80 (n=17) or 160 μM (n=10) hydrogen peroxide for 10 min, followed by 10 min recovery, before 25 min global ischaemia and 60 min reperfusion, and compared with ischaemic controls of matching perfusion time (n=17 and n=23). Cardiac performance was assessed by heart rate, left ventricular systolic, end-diastolic and developed pressures, and coronary flow. Severe reperfusion arrhythmias occurred frequently in control hearts, and was attenuated by ischaemic preconditioning. All hearts pretreated with 160 μM hydrogen peroxide had severe arrhythmias throughout reperfusion, while these were not seen in any heart perfused with 20 μM hydrogen peroxide (P< 0.01 compared to controls). Ischaemia and reperfusion induced a minor decrease in heart rate, left ventricular systolic and developed pressures, and increased end-diastolic pressure. Ischaemic preconditioning attenuated the decrease of heart rate and the increase of end-diastolic pressure, and increased coronary flow, while hydrogen peroxide did not significantly attenuate these changes. In conclusion, a low dose of exogenous hydrogen peroxide before global ischaemia inhibited severe reperfusion arrhythmias, but had no other protective effects. The present work does not suggest that reactive oxygen species are important mediators of the preconditioning effects on stunning and arrhythmias in the rat heart.     

The combination of L-arginine and ischaemic post-conditioning at the onset of reperfusion limits myocardial injury in the pig

Aim: To investigate whether ischaemic post‐conditioning (IPoC) combined with i.v. infusion of the nitric oxide (NO) substrate l ‐arginine at the onset of reperfusion exerts cardioprotective effect that is superior to either treatment given separately. Methods: Twenty‐six anesthetized pigs were subjected to coronary artery (left anterior descending artery, LAD) ligation for 40 min followed by 4 h reperfusion. The pigs were randomized into five different groups receiving either i.v. vehicle, i.v. l ‐arginine, IPoC 4 × 60 s together with i.v. vehicle or IPoC together with i.v. l ‐arginine and a group with IPoC 8 × 30 s. All infusions were started 10 min before reperfusion. Results: The infarct size of the vehicle group was 82 ± 4% of the area at risk. l ‐Arginine alone (79 ± 8%), IPoC 4 × 60 s vehicle (86 ± 3%) or IPoC 8 × 30 s vehicle (94 ± 7%) did not affect infarct size. l ‐Arginine together with IPoC significantly reduced infarct size to 59 ± 4% (P < 0.01). Except for higher LAD flow during early reperfusion in the IPoC l ‐arginine group, haemodynamic parameters did not differ between the four main groups. Heart rate and rate pressure product were lower during ischaemia and reperfusion in the IPoC 8 × 30 s vehicle group. In comparison with the vehicle group, there were no changes in the expression of Akt, phosphorylated Akt Ser⁴⁷³, inducible NO synthase, endothelial NO synthase (eNOS) or phosphorylated eNOS Ser¹¹⁷⁷ in the ischaemic/reperfused myocardium. Conclusion: l ‐Arginine given systemically at the onset of reperfusion protects the pig heart against ischaemia and reperfusion injury only when combined with IPoC. These results indicate that the combination of the two treatment strategies exerts cardioprotection.     

Post‐ischaemic activation of kinases in the pre‐conditioning‐like cardioprotective effect of the platelet‐activating factor

Aim: Platelet‐activating factor (PAF) triggers cardiac pre‐conditioning against ischemia/reperfusion injury. The actual protection of ischaemic pre‐conditioning occurs in the reperfusion phase. Therefore, we studied in this phase the kinases involved in PAF‐induced pre‐conditioning. Methods: Langendorff‐perfused rat hearts underwent 30 min of ischaemia and 2 h of reperfusion (group 1, control). Before ischaemia, group 2 hearts were perfused for 19 min with PAF (2 × 10^?11 m ); groups 3–5 hearts were co‐infused during the initial 20 min of reperfusion, with the protein kinase C (PKC) inhibitor chelerythrine (5 × 10^?6 m ) or the phosphoinositide 3‐kinase (PI3K) inhibitor LY294002 (5 × 10^?5 m ) and atractyloside (2 × 10^?5 m ), a mitochondrial permeability transition pore (mPTP) opener respectively. Phosphorylation of PKCε, PKB/Aκt, GSK‐3β and ERK1/2 at the beginning of reperfusion was also checked. Left ventricular pressure and infarct size were determined. Results: PAF pre‐treatment reduced infarct size (33 ± 4% vs. 64 ± 5% of the area at risk of control hearts) and improved pressure recovery. PAF pre‐treatment enhanced the phosphorylation/activation of PKCε, PKB/Aκt and the phosphorylation/inactivation of GSK‐3β at reperfusion. Effects on ERK1/2 phosphorylation were not consistent. Infarct‐sparing effect and post‐ischaemic functional improvement induced by PAF pre‐treatment were abolished by post‐ischaemic infusion of either chelerythrine, LY294002 or atractyloside. Conclusions: The cardioprotective effect exerted by PAF pre‐treatment involves activation of PKC and PI3K in post‐ischaemic phases and might be mediated by the prevention of mPTP opening in reperfusion via GSK‐3β inactivation.     

Involvement of endogenous adenosine in ischaemic preconditioning in swine

Adenosine release and the subsequent activation of adenosine receptors are involved in ischaemic preconditioning in dogs and rabbits. In the present study, we investigated whether adenosine also mediates ischaemic preconditioning in swine. Swine were used since, due to the lack of an innate collateral circulation, infarct development in this species most closely resembles that observed in humans. In 36 enflurane-anaesthetized swine the impact of increased adenosine breakdown with exogenous porcine adenosine deaminase (5 IU/ml blood/min) on global and regional myocardial function (sonomicrometry), subendocardial blood flow (ENDO, microspheres) and infarct size (IS, triphenyl tetrazolium chloride staining following 90 min ischaemia and 120 min reperfusion) were analysed. Low-flow ischaemia for 90 min at an ENDO of 0.09±0.04 (mean±SD) ml/min/g caused an IS of 13.2±9.7% (n=8) of the area at risk. Ischaemic preconditioning by a cycle of 10 min low-flow ischaemia followed by 15 min reperfusion prior to the 90-min ischaemic period (ENDO=0.06±0.03 ml/min/g) reduced IS to 2.6±3.0% (n=11, P<0.05). The interstitial adenosine concentration (microdialysis) increased from 1.60±0.87 nmol/ml to above 10 M during ischaemia; with intracoronary adenosine deaminase, the interstitial adenosine concentration fell from 1.65±0.23 to 0.12±0.07 nmol/ml and did not increase during ischaemia. Adenosine deaminase per se did not alter IS after 90 min ischaemia (n=7, ENDO=0.08±0.04 ml/min/g, IS=12.1±6.9%) but abolished the beneficial effect of ischaemic preconditioning (n=10, ENDO=0.06±0.03 ml/min/g, IS=8.8±5.8%). For any given ENDO, IS was significantly reduced in the ischaemic preconditioned group compared with the other three groups. Global and regional myocardial function were comparable among all groups of swine. We conclude that endogenous adenosine mediates ischaemic preconditioning also in swine.     

Preconditioning does not attenuate cardiac dysfunction after global ischaemia in the guinea-pig

Ischaemic preconditioning reduces infarct size, but the effects on cardiac function after global ischaemia are more controversial. Additionally, species differences may exist. The present study investigates the effects of preconditioning on cardiac performance in the globally ischaemic, Langendorff-perfused guinea-pig heart. Hearts were stabilized for 25 min, and divided into the following groups: (1) (n = 8) control perfusion for 16 min before 30-min global ischaemia and 30-min reperfusion, (2) (n = 7) two episodes of 3-min ischaemia and 5-min reperfusion before global ischaemia, (3) (n = 7) 5-min ischaemia and 10-min reperfusion before ischaemia, (4) (n = 8) control perfusion before 40-min ischaemia and 30-min reperfusion, (5) (n = 8) Preconditioning as group 2 before ischaemia as group 4, (6) (n = 9) Control perfusion before 50-min ischaemia and 30-min reperfusion, (7) (n = 10) Preconditioning as group 2 before ischaemia as group 6. A dose-dependent reduction of left ventricular systolic pressure, and increase of end-diastolic pressure was observed during reperfusion after 30-, 40- and 50-min ischaemia. Preconditioning did not influence these changes, nor did it attenuate the incidence of severe reperfusion arrhythmias or reduction of coronary flow. In conclusion, ischaemic preconditioning does not improve cardiac function during reperfusion of the globally ischaemic, isolated guinea-pig heart.     

Various inotropic effects of angiotensin II in post‐ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C

Aims: The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post‐ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. Methods: Isolated Langendorff‐perfused rat hearts were perfused under constant flow with oxygenated Krebs–Henseleit buffer and paced at 360 beats min^?1. A saline‐filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15‐ or 25‐min global ischaemia followed by 45‐min reperfusion. At the end of reperfusion, 0.1 μmol L^?1 AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25‐min ischaemia followed by 45‐min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 μmol L^?1). Results: The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P < 0.05 vs. prior to AngII infusion). In post‐ischaemic hearts subjected to 15‐min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P < 0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. Conclusions: AngII exerts negative inotropic effect in severely injured post‐ischaemic heart, possibly through the PKC pathway.     

Relationship between ischaemic time and ischaemia/reperfusion injury in isolated Langendorff‐perfused mouse hearts

Myocardial functional recovery and creatine kinase (CK) release following various periods of ischaemia were investigated in isolated mouse hearts. The hearts were perfused in the Langendorff mode with pyruvate‐containing Krebs–Hensleit (KH) buffer under a constant perfusion pressure of 80 mmHg, and were subjected to either continuous perfusion or to 5, 15, 20, 25, 30, 45 or 60 min of global ischaemia followed by 45 min of reperfusion. In hearts subjected to ischaemic periods of 5, 15 or 20 min, there was a transient reduction in the left ventricular (LV) dP/dt max during the early phase of reperfusion, while the recovery at the end of reperfusion reached a level similar to that in hearts subjected to continuous perfusion. In hearts subjected to longer ischaemic periods, i.e. 25, 30, 45 or 60 min, the decrease in the cardiac performance was more pronounced and persistent, with significantly lower recovery in LV dP/dt max and higher LV end diastolic pressure (LVEDP) at the end of reperfusion than in the non‐ischaemic hearts. There were no significant differences in the recoveries in coronary flow or in heart rate (HR) between groups. Similarly to the functional recovery, the release of CK showed a clear ischaemic length‐related increase. In conclusion, the Langendorff‐perfused isolated mouse heart could be a valuable model for studies of myocardial ischaemia/reperfusion injury. Future studies using gene‐targeted mice would add valuable knowledge to the understanding of myocardial ischaemia/reperfusion injury.     

Oral pre‐treatment with rosuvastatin protects porcine myocardium from ischaemia/reperfusion injury via a mechanism related to nitric oxide but not to serum cholesterol level

Aims: The aim of this study was to test whether oral pre‐treatment with rosuvastatin at a dosage giving clinically relevant plasma concentrations protects the myocardium against ischaemia/reperfusion injury and to investigate the involvement of nitric oxide (NO) and neutrophil infiltration. Methods: Pigs were given placebo (n = 7), rosuvastatin (80 mg day^?1, n =7), rosuvastatin (160 mg day^?1, n = 7) or pravastatin (160 mg day^?1, n = 7) orally for 5 days before being subjected to coronary artery ligation and reperfusion. An additional group was given rosuvastatin 160 mg day^?1 and a nitric oxide synthase (NOS) inhibitor. Results: Rosuvastatin 80 and 160 mg day^?1 resulted in plasma concentrations of 2.6 ± 0.7 and 5.6 ± 1.0 ng mL^?1, respectively. Serum cholesterol was not affected. Rosuvastatin 160 mg day^?1 and pravastatin limited the infarct size from 82 ± 3% of the area at risk in the placebo group to 61 ± 3% (P < 0.05), and to 61 ± 2% (P < 0.05) respectively. Rosuvastatin 80 mg day^?1 limited the infarct size to 69 ± 2%, however, this effect was not statistically significant. Rosuvastatin 160 mg day^?1 attenuated neutrophil infiltration in the ischaemic/reperfused myocardium. The protective effect of rosuvastatin 160 mg day^?1 was abolished by NOS inhibition. The expression of NOS2 and NOS3 in the myocardium did not differ between the groups. Conclusions: Oral pre‐treatment with rosuvastatin limited infarct size following ischaemia/reperfusion without affecting cholesterol levels. The cardioprotective effect is suggested to be dependent on maintained bioactivity of NO, without influencing NOS expression.     

Measurement of local blood flow in acute myocardial infarction: loss of 15-^m microspheres during the first hour

Distribution of radiolabeled microspheres is widely utilized for determination of regional blood flow in experimental myocardial infarction studies. The purpose of this investigation was evaluation of the microsphere method during 1 h of regional ischaemia. Special attention was focused upon loss of preocclusion microspheres from ischaemic myocardium; mechanisms for loss and blood flow distribution in non-ischaemic left ventricle. Microspheres (15 μm) were injected into the left atrium in nine pentobarbital anaesthetized cats prior to coronary artery occlusion and again after 1 h of occlusion. Preocclusion blood flow estimates were lower in ischaemic compared with non-ischaemic myocardium (1.36 us. 1.62 cm³min^-1 g^-1, P = 0.002), corresponding to 16%apparent loss. In endocardial ischaemic tissue, development of oedema could account for the loss. In epicardial ischaemic tissue, oedema was not present and loss was therefore due to migration of microspheres. Epicardial loss increased in proportion to restoration of left ventricular contractility. There was no evidence for significant microsphere loss through lymphatic pathways. In non-ischaemic left ventricular tissue, myocardial blood flow was evenly distributed from apex to base, and also between endocardial and epicardial layers. This study quantitates an important limitation to measurements of local blood flow in ischaemic myocardium by radiolabelled microspheres.     

The effect of a synthetic hexadentate iron chelator (CP130) and desferrioxamine on rabbit kidneys exposed to cold and warm ischaemia

The effect of CP130 (a synthetic hexadentate pyridinone iron chelator) on the formation of two markers of lipid peroxidation (TBA-reactive material and Schiff's bases) in rabbit kidneys following a 72 h period of cold (0–4°C) ischaemia was investigated by either adding CP130 to the flush/storage solution (hypertonic citrate solution) or by administering the agent intravenously 15 min before removal of the organs. In both cases, CP130 blocked the adverse rises in lipid peroxidation caused by ischaemia and subsequent reoxygenation of the homogenatesin vitro. Both CP130 and desferrioxamine (DFX) (administered intravenously 15 min before ischaemia and 5 min before reperfusion) were also found to significantly reduce post-ischaemic rates ofin vitro lipid peroxidation in kidneys rendered warm ischaemic for 90 min followed by reperfusion for 5 or 60 minin situ. Kidneys exposed to warm ischaemia and reperfusion developed interstitial and intracellular oedema, congestion and haemorrhage. DFX administration had little effect on the histological outcome, whereas CP130 significantly reduced interstitial oedema (at 6 min reperfusion compared to the DFX-treated group), intracellular oedema (at 60 min reperfusion compared to the DFX-treated group) and congestion (at 5 min reperfusion compared with a control group not given any agent). It is concluded that while CP130 and DFX exhibited similar antioxidant properties, CP130 provided better protection from ischaemia/reperfusion injury at the histological level. Synthetic iron chelators may therefore be of benefit in clinical organ transplantation by protecting against tissue damage caused by prolonged ischaemia.     

Modulation of nitric oxide affects myocardial perfusion-contraction matching in anaesthetized dogs with recurrent no-flow ischaemia

Myocardial perfusion and contraction are closely coupled; however, the effect of recurrent no-flow ischaemia on perfusion-contraction matching remains to be established. In the present studies, we examined the influence of modulating nitric oxide availability on perfusion-contraction matching after recurrent no-flow ischaemia in acute open-chest, anaesthetized dogs. The following three groups were studied: (1) saline; (2) L-NAME (10 mg kg(-1) I.V.); and (3) enalaprilat (1.5 mg kg(-1) I.V.). Regional myocardial blood flow was measured with microspheres and contractile function with piezoelectric crystals to determine systolic wall thickening. Dogs underwent four cycles of 5 min acute ischaemia and 5 min coronary reperfusion; area at risk was similar for all groups. In all dogs, ischaemic zone contractile function was depressed after recurrent no-flow ischaemia despite increased myocardial blood flow during reperfusion; contractile function was further depressed during L-NAME and was partly restored with enalaprilat. Within the ischaemic region, blood flow in subendocardial and subepicardial layers increased significantly compared with baseline during each reperfusion period independently of treatment. Our findings suggest that reduced NO availability can significantly impair myocardial perfusion-contraction matching, which is partly restored by administration of an NO donor.     

Regional myocardial heat-shock protein (HSP70) concentrations under different blood flow conditions

 The concentration of heat-shock proteins of 70 kD (HSP70) in heart tissue has been shown to increase during transient myocardial ischaemia and to persist during several hours of reperfusion. In this study the relationship between the local myocardial HSP70 concentration and blood flow was addressed for control physiological conditions and acute myocardial ischaemia. A specific aim of this study was to address the question of whether low flow areas under control physiological conditions have undergone a transient ischaemia during the preceding hours and thus may be in a state of hibernation or stunning. In 12 anaesthetized, open-chest beagle dogs (6 control and 6 with 60-min coronary artery stenosis) heart rate, mean aortic pressure, mean arterial partial pressure of O₂ and partial pressure of CO₂ averaged 85±16 beats/min, 94±14 mmHg, 102±17 mmHg and 39±6 mmHg, respectively. Regional HSP70 and myocardial blood flow (RMBF) were measured using an HSP70-enzyme-linked immunosorbent assay and the tracer microsphere technique, respectively, in samples of 250 mg wet mass. In the control group the mean RMBF was 1.06±0.59 ml·min^–1·g^–1 and the local HSP70 concentration was 7.08±1.03 μg/mg cytosolic protein. Myocardial HSP70 showed a blood flow-independent regional biological heterogeneity, equivalent to a coefficient of variation of 0.31. Local HSP70 concentrations did not differ (P>0.05) between control low and high flow samples, 6.16±1.0 vs 6.08±0.75 μg/mg cytosolic protein, respectively. However, after 60 min of coronary artery occlusion the local HSP70 concentration increased from 7.08 ±1.03 to 13.43±3.19 μg/mg cytosolic protein (P<0.001). There was a significant inverse relationship between the percent reduction of local blood flow and HSP70 (r=–0.56, P<0.001). From these results it is concluded that: (1) low flow samples under control physiological conditions are unlikely to be in a state of hibernation or stunning since their HSP70 concentration is normal and (2) the increase in the local HSP70 concentration during myocardial ischaemia reflects the degree of impairment of O₂ delivery. Received: 29 May 1998 / Received after revision: 14 August 1998 / Accepted: 25 August 1998     

Contractile properties of skinned preparations from ischaemic canine myocardium and coronary arteries

The influence of prolonged ischaemia on the regulation of contraction in the myocardium and in the smooth muscle of coronary arteries was investigated. Chemically skinned preparations were used which enabled the contraction to be studied with the environment of the contractile filaments controlled. Myocardial ischaemia was produced in anaesthetized adult beagle dogs by occlusion of the left anterior descending artery for 3 h and followed by 30 min reperfusion. Myocardial tissue and segments from coronary arteries were obtained from the ischaemic infarcted wall region (in vivo ischaemic) and compared with control preparations from perfused coronary arteries and from the free wall of the left ventricle. Coronary and myocardial preparations were also obtained from the heart after a 3 h period in vitro under anoxic conditions at 37°C (in vitro ischaemic) simulating a state of extreme ischaemia. Control myocardial fibres were fully relaxed at pCa (-log-[Ca²⁺]) 9 and developed 24±5% (n=7) of maximum force at intermediate calcium concentration (pCa 5.5). In contrast, the in vivo and in vitro ischaemic preparations produced force at pCa 9 (28±13 and 39±8%, respectively, n=5 and 7) and showed an increased force development at pCa 5.5 (53±11 and 75±5%). The in vivo and in vitro ischaemic coronary arteries relaxed more slowly following calcium removal than control vessels. The in vitro ischaemic vascular preparations developed active force at pCa 9 and showed increased levels of myosin light chain phosphorylation and reduced phosphatase activity. This suggests a reduced rate of dephosphorylation as a cause for the changes in contracile behaviour of the smooth muscle. In conclusion, extreme ischaemia in vitro is associated with a loss of calcium regulation and an increased calcium sensitivity of the contractile system in myocardium and changes in the phosphorylation/dephosphorylation reactions of coronary arteries. The changes in myocardium appear to occur also during ischaemia in vivo, and might contribute to contracture development in cells under conditions when adenosine triphosphate synthesis is reestablished after reperfusion.     

Characterization of the cortical laser-Doppler flow and hippocampal degenerative patterns after global cerebral ischaemia in the goat

 Large-animal models offer several advantages in the study of cerebral ischaemia: easier control of physiological variables, easier neuropathological evaluation, etc. In the present study we have taken advantage of the unique cerebrovascular anatomy of the goat to reproduce a model of reversible, incomplete, global cerebral ischaemia in a large-sized animal species, in which the effects of successive manoeuvres to stop and re-start cerebral blood flow can be recorded continuously. Early cortical laser-Doppler flow response (up to 2 h) and delayed neuronal degeneration (7 days) in the hippocampal CA1 subfield have been analysed in goats undergoing 5, 10 or 20 min of transient, global cerebral ischaemia. Bilateral occlusion of the external carotid artery plus compression of jugular veins reduced cortical laser-Doppler flow to 11 ± 8% of preischaemic values (P<0.01), flattened the electrocorticogram, and increased mean arterial blood pressure by 17 ± 23% (P<0.01) and intracranial pressure by 161 ± 136% (P<0.01). A rather heterogeneous response was obtained during reperfusion: 14 out of 31 goats showed the ”classical” pattern consisting of hyperaemia followed by delayed hypoperfusion. The remaining goats showed neither hyperaemia (11 goats) nor delayed hypoperfusion (6 goats). The duration of the ischaemic insult did not correlate with the magnitude of hyperaemia or delayed hypoperfusion, but influenced neurodegeneration: while no loss of hippocampal CA1 neurons was observed at 7 days after 5 or 10 min ischaemia, a 68% cell loss was observed in the 20-min ischaemia group. Our goat model has thus proven to be very suitable for the induction of global cerebral ischaemia in a large-animal species without extensive surgery. It allows reproducible reductions of cerebral blood flow, long-term recovery, low mortality rate, and high incidence of neuronal damage. The results reported here support the view that delayed hypoperfusion is not an important determinant of neuronal injury. Received: 23 September 1997 / Received after revision and accepted: 3 December 1997