Short Periods Of Ischemia Research Articles

Role and mechanisms of secondary mitochondrial failure.

Ischemia is accompanied by mitochondrial dysfunction, as assessed by measurements of mitochondrial respiratory activities in vitro. Following brief periods of ischemia, mitochondrial function is usually normalized during reperfusion. However, particularly after ischemia of longer duration, reperfusion may be accompanied by secondary mitochondrial failure. After short periods of ischemia this is observed in selectively vulnerable areas and, after intermediate to long periods of ischemia, in other areas as well. However, it has remained unsettled if the mitochondrial dysfunction is the result or the cause of cell death. Although it has been commonly assumed that such failure is secondary to cell injury by other mechanisms, recent results suggest that mitochondrial dysfunction may be the cause of cell death. Indirect evidence for this postulate is provided by experiments showing that cyclosporin A (CsA), when allowed to cross the blood-brain barrier, is a potent neuroprotectant. CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore, a voltage-gated channel allowing molecules and ions with a mass < 1500 Daltons to pass the inner mitochondrial membrane. Experiments on isolated cells in vitro demonstrate that cell calcium accumulation or oxidative stress triggers the assembly of an MPT pore, which leads to collapse of the mitochondrial membrane potential, to ATP hydrolysis, to enhanced production of reactive oxygen species (ROS), and to cell death. The beneficial effect of CsA could thus be related to its ability to block the MPT pore. Longer periods of ischemia, such as occurs after transient middle cerebral artery (MCA) occlusion, lead to pan-necrotic lesions (infarction). In the rat, recirculation following 2 h of MCA occlusion leads to partial normalization of the bioenergetic state but this is followed within 4-6 h by secondary bioenergetic failure. The latter seems unrelated to blockade of the microcirculation, but correlates to secondary mitochondrial failure. The brain damage incurred is ameliorated by the spin trap alpha-phenyl-N-butyl nitrone (PBN) and by the immunosuppressant FK506 even when given 1-3 h after the start of recirculation. The two drugs also prevent the secondary mitochondrial failure during early recirculation, suggesting that such failure is pathogenetically important. Probably, though, the mitochondrial dysfunction involves not only the assembly of an MPT pore but also other mechanisms. Since recirculation is associated with release of mitochondrial proteins it is not unlikely that such proteins, e.g. cytochrome c, trigger cascades of events leading to cell death.6.

Advantages of continuous hyperpolarized arrest with pinacidil over St. Thomas' hospital solution during prolonged ischemia

Objective: The potassium channel opener, pinacidil, has been shown to be an effective cardioplegic agent over short periods of ischemia. However, clinical settings typically involve longer ischemic periods. This study tested the hypothesis that myocardial protection with a potassium channel opener is feasible during prolonged periods of arrest and is comparable with the traditional St. Thomas' Hospital solution. Methods: With a blood-perfused, isolated rabbit heart model, hearts underwent 1 hour of global normothermic ischemia followed by 30 minutes of reperfusion. During ischemia, three different cardioplegic solutions were administered either intermittently by infusion every 20 minutes or as continuous low-flow cardioplegia (150 ml total volume in all groups): (1) Krebs-Henseleit solution alone (control), (2) Krebs-Henseleit solution + pinacidil (50 μmol/L), or (3) St. Thomas' Hospital solution. Initial potassium channel opener infusions contained 5 mmol/L procaine. Postreperfusion systolic function (percent of developed pressure) was measured. Compliance changes were integrated from the end-diastolic pressure/volume relationships. Results: For intermittent cardioplegia, only St. Thomas' Hospital solution improved function (62.5% ± 4.0%) versus control (43.6% ± 3.3%, p < 0.001). However, with continuous cardioplegia, only pinacidil (75.6% ± 4.8%) exceeded control (62.7% ± 2.2%, p < 0.001) and was significantly better than St. Thomas' Hospital solution. Compared with the intermittent control group, all other groups showed significant preservation of preischemic diastolic properties. Conclusions: Myocardial protection during a longer, more clinically relevant ischemic period is feasible with a potassium channel opener only when it is given continuously. Continuous low-flow pinacidil cardioplegia was superior to St. Thomas' Hospital solution given either as an intermittent or continuous infusion. (J Thorac Cardiovasc Surg 1998;116:131-8)

Upregulation of the Enzyme Chain Hydrolyzing Extracellular ATP after Transient Forebrain Ischemia in the Rat

A short ischemic period induced by the transient occlusion of major brain arteries induces neuronal damage in selectively vulnerable regions of the hippocampus. Adenosine is considered to be one of the major neuroprotective substances produced in the ischemic brain. It can be released from damaged cells, but it also could be generated extracellularly from released ATP via a surface-located enzyme chain. Using the rat model of global forebrain ischemia, we applied a short (10 min) transient interruption of blood flow and studied the distribution of ectonucleotidase activities in the hippocampus. Northern hybridization of mRNA isolated from hippocampi of sham-operated and ischemic animals revealed an upregulation of ectoapyrase (capable of hydrolyzing nucleoside 5'-tri- and diphosphates) and ecto-5'-nucleotidase (capable of hydrolyzing nucleoside 5'-monophosphates). A histochemical analysis that used ATP, UTP, ADP, or AMP as substrates revealed a strong and selective increase in enzyme activity in the injured areas of the hippocampus. Enhanced staining could be observed first at 2 d. Staining increased within the next days and persisted at 28 d after ischemia. The spatiotemporal development of catalytic activities was identical for all substrates. It was most pronounced in the CA1 subfield and also could be detected in the dentate hilus and to a marginal extent in CA3. The histochemical staining corresponded closely to the development of markers for reactive glia, in particular of microglia. The upregulation of ectonucleotidase activities implies increased nucleotide release from the damaged tissue and could play a role in the postischemic control of nucleotide-mediated cellular responses.

Ischemia and Ischemic Preconditioning in the Buffer-Perfused Pigeon Heart

Isolated pigeon hearts were perfused with Krebs-Henseleit bicarbonate buffer with 1.25 mM Ca ++ at a pressure of 60 cm H 2O and paced at 210 beats per min. After an equilibration perfusion of 30 min, hearts were subjected to 10 min global ischemia and then reperfused for 30 min. Left ventricular +dP/dt max, systolic, and end diastolic pressures differed significantly from baseline values during reperfusion as did the release of lactate dehydrogenase (LDH). When the hearts were preconditioned by interruption of flow for two 2.5-min intervals, followed by 10 min of ischemia and then reperfusion, the short periods of ischemia, followed by reperfusion, protected the hearts against the longer bout of ischemia as evidenced by significant differences between the left ventricular (LV) pressure, +dP/dt max, LV end diastolic pressure and LDH values obtained from the hearts of control vs. preconditioned hearts. Substitution of 1 μM adenosine for the preconditioning ischemia also resulted in the preconditioning response. Ischemic preconditioning (IP) was not blocked by addition of 100 μM 8-(- p-sulfophenyl) theophylline, an adenosine receptor antagonist. Therefore, isolated, perfused bird hearts can be preconditioned, and the mechanism may involve adenosine receptors, although their activation is not necessary for IP to occur. Factors in addition to adenosine are likely involved.

Nitric oxide enhances endothelin production in pancreas transplantation.

The role of endothelin and its relationship with nitric oxide (NO) production in ischemia-reperfusion associated with pancreas transplantation has been explored. For this purpose, pancreatic levels of endothelin were evaluated in an experimental model of pancreas transplantation after different periods of cold preservation. The effects of NO synthase inhibition were also evaluated. Results show posttransplantation increases in lipase and endothelin production. The release of lipase and endothelin was only prevented by NG-nitro-L-arginine methyl ester after a short ischemic period. Thus, endothelin synthesis could be a consequence of stimulation with NO in the ischemia-reperfusion associated with pancreas transplantation.

Effects of pentoxifylline on neutrophil function in patients with intermittent claudication

Short periods of ischemia, as in intermittent claudication, cause alterations in neutrophil activation and deformability, which may impair the microcirculation. The aim of this study was to evaluate the effect of pentoxifylline (PTX) on neutrophil function in patients with unilateral intermittent claudication. In 15 patients with predominantly unilateral peripheral arterial occlusive disease, lactate, proportion of activated neutrophils (by nitro blue tetrazolium test), neutrophil filterability (by St. George's filtrometer), and stimulated as well as unstimulated radical generation were assessed in the femoral venous blood of the diseased and healthy leg. The values were obtained at rest, immediately after claudication, and 15 minutes after claudication. After 14 days of treatment with PTX (3 × 400 mg/die p.o.) the measurements were repeated. Pentoxifylline reduced the effects of ischemia on neutrophil function, whereas the same exercise level before and after treatment was reached as measured by lactate production. Ischemia induced elevation in the proportion of activated neutrophils (+43%, p < 0.05) and in unstimulated radical generation (+10.7%, p < 0.05) was diminished by treatment with pentoxifylline (+2.4%, +1.9%, respectively). Decrease in filterability of neutrophils due to exercise was reduced from −17.5% to −12.4% (p < 0.05) and stimulated radical generation decreased from +19.8% to +6.4% (p < 0.05). In conclusion, PTX inhibits ischemia-induced alteration in neutrophil function in patients with intermittent claudication.

Differences in Reperfusion Length Following 30 Minutes of Ischemia in the Rabbit Influence Infarct Size, as Measured by Triphenyltetrazolium Chloride Staining

Assessment of myocardial infarct size in acute experimental models is usually done by triphenyltetrazolium-chloride (TTC) staining. A certain period of reperfusion is mandatory for discrimination of the infarct zone, especially after relatively short ischemic periods. However, it is unclear what the optimal reperfusion time is for full delineation of the infarct following 30 min of myocardial ischemia in the rabbit. This study compares infarct size, assessed by TTC, in anesthetized open-chest rabbits subjected to 30 min of coronary artery occlusion followed by either 2 (n=14)v4 (n=14) (protocol 1), or 3 (n=8)v6 (n=7) h of reperfusion (protocol 2). Area at risk was assessed by blue dye and necrotic zone by TTC staining. Protocol 1: heart rate and mean blood pressure were comparable in both groups throughout the protocol. Regional myocardial blood flows in both the ischemic and non-ischemic zones during ischemia and after 2 h of reperfusion were comparable between the groups. Regional myocardial blood flow in the post-ischemic zone deteriorated between 2 and 4 h (1.11±0.15v0.58±0.09 ml/min/g, respectively;P=0.0004) of reperfusion. The size of the area at risk was comparable (0.31±0.03v0.33±0.03 of the LV weight in the 2 and 4 h reperfusion groups). However, the ratio of the necrotic zone to the ischemic zone at risk was 63% larger in the 4 compared to the 2 h of reperfusion group (0.31±0.04v0.19±0.03, respectively,P=0.02). Analysis of covariance performed on the weight of tissue that developed necrosis and the weight of ischemic zone at risk revealed a significant effect of the reperfusion time (P=0.014). Protocol 2: there was no difference in infarct size between rabbits subjected to three (0.38±0.05 of the area at risk)v6 h (0.41±0.07) of reperfusion (P=0.72). Analysis of covariance performed on the weight of tissue that developed necrosis and the weight of ischemic zone at risk did not reveal a significant effect of the reperfusion time. Infarct size as assessed by TTC following 30 min of myocardial ischemia, is smaller when measured 2 h after reperfusion than after 4 h of reperfusion. At least 3 h of reperfusion is needed to delineate infarct size by tetrazolium staining following 30 min of ischemia.

Myocardial functional preservation during ischemia: influence of beta blocking agents.

To determine whether prior acute Beta blockade protects the heart against the deleterious effects of normothermic low flow global ischemia on myocardial function, aortic pressure, developed pressure, dP/dtmax and end diastolic pressure were monitored in isolated perfused rabbit hearts prior to, during and following 30 and 60 min ischemia, during which either Krebs-Henseleit (control) or Beta blocking agents. Bevantolol (cardioselective) or Propranolol (non-selective) were perfused through the heart. Control hearts made ischemic for 30 min and then reperfused had significantly elevated end diastolic (p < .01) and aortic pressures (p < .01) and reduced developed pressure relative to baseline (p < .05). Hearts treated with Bevantolol or Propranolol (3 x 10(-5) m/l) 5 min prior to and during 30 min ischemia recovered preischemic developed pressure and dP/dtmax (p > 0.05), while end diastolic pressure was elevated (p < .01, p < .05 respectively). Aortic pressure was unchanged relative to baseline (p > .05). Comparison of indices from hearts under Beta blockade with controls showed that following 30 min ischemia and recovery, the Bevantolol treated group had reduced aortic pressure (p < .01) and end diastolic pressure (p < .05) and increased percent developed pressure and percent dP/dtmax (p < .001) relative to control. In the propranolol treated group, end diastolic pressure was reduced and percent developed pressure (p < .01) and percent dP/dtmax (p < .001) were increased relative to unblocked hearts. Following 60 min ischemia and 30 min reperfusion, reduction in all functional indices occurred, however dP/dtmax was unchanged from baseline in the Propranolol and Bevantolol treated groups. Comparison between groups showed that the Bevantolol treated group had significantly better dP/dtmax and developed pressure (p < .05), whereas the Propranolol group shows no significant difference from baseline (p > .05) (K-H). We conclude that following short periods of ischemia, Beta blockade protects the heart from deleterious function effects of ischemia but that the protective effect is diminished in Bevantolol relative to Propranolol treatments following prolonged ischemia. The data indicates that the beneficial effects of Beta blockade in reducing ischemic induced damage occurs early during conditions of ischemia such as would be present in the setting of acute myocardial infarction.

Enterocyte viability and mitochondrial function after graded intestinal ischemia and reperfusion in rats.

Ischemia/reperfusion of the small intestine can lead to metabolic and structural alterations in the mucosa. Cellular dysfunction occurs when mitochondrial metabolism is compromised, which may ultimately lead to impaired organ function. The aims of this study were to assess the suppression of cellular and mitochondrial oxidative metabolism and involvement of mitochondria in the ischemia/reperfusion injury. The mitochondria were prepared from isolated enterocytes obtained from the small intestine of anesthetized adult rats following different time periods of ischemia and ischemia followed by 5 min reperfusion. Cellular and mitochondrial function were assessed using MTT (3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) reduction assay. Ischemia of increasing time periods caused a progressive decrease in cellular and mitochondrial MTT reduction in enterocytes and reperfusion showed further decrease of MTT formazan formation. Inclusion of 1 mM succinate, as respiratory substrate, showed reversal of suppression of mitochondrial function in 30-60 min ischemia whereas 90 min ischemia or short time period ischemia followed by 5 min reperfusion indicated an irreversible damage to mitochondria. This study indicated that mitochondria are a sensitive target of damage due to oxygen deficiency and possibly due to sudden burst of oxygen free radicals. Mitochondria can withstand short periods of ischemia whereas long duration ischemia or reperfusion results in irreversible damage to mitochondrial function.

Prolonged coronary artery occlusion—reperfusion sequences reduce myocardial free radical production: An electron paramagnetic resonance study

Our purpose was to determine whether prolonged myocardial ischemia attenuates free radical production after early reperfusion. Twenty-two mongrel dogs underwent left anterior descending coronary artery occlusion for 20, 40, or 60 minutes followed by 30 minutes of reperfusion. Electron paramagnetic resonance spectroscopy was used to measure ascorbate free radical in the coronary vein effluent. Ascorbate free radical production during reperfusion was significantly ( p < 0.05) reduced in the dogs undergoing 60 minutes of coronary artery occlusion compared with the dogs undergoing 40 and 20 minutes of occlusion. We conclude that prolonged myocardial ischemia results in less free radical production on reperfusion than do shorter periods of ischemia followed by reperfusion.

Alterations in the glutathione content of mitochondria following short-term forebrain ischemia in rats

Total glutathione was measured in mitochondria isolated following 30 min of ischemia and recirculation periods up to 24 h. Mitochondria prepared from the dorsolateral striatum, a region containing many neurons susceptible to short ischemic periods, were compared with those from the paramedian cortex, an ischemia-resistant region. Parallel increases in glutathione content (to approximately 150% of pre-ischemic values) were seen in both regions during the first few hours of recirculation. By 24 h of recirculation, there was a decrease below pre-ischemic values in preparations from the dorsolateral striatum but not the paramedian cortex. The early increases in mitochondrial glutathione were not associated with comparable increases in total tissue glutathione. A shorter (10 min) ischemic period also produced an early increase in mitochondrial glutathione but this was reversed more rapidly to preischemic values. The observed changes indicate post-ischemic modifications of cellular oxidative defences in the two brain regions studied.

Molecular motor mechanics in the contracting heart. V1 versus V3 myosin heavy chain.

The amount of iron in the low molecular weight pool (LMW) increases during no-flow ischemia and is thought to be essential to oxygen radical-derived damage upon reperfusion. Applying three short ischemic periods (5 min) preconditioning before 15 min ischemia results in an improved contractility compared to a direct 15 min ischemic insult. This raises the question whether preconditioning leads to a decrease in hte LMW iron pool. We therefore investigated the change in in hte LMW iron pool during ischemic insult after applying preconditioning. It is assumed that an increase in LMW iron is dependent on the accumulation of reduction equivalents derived from the anaerobic glycolysis. Therefore the glycogen content was also reduced by administration by anoxia and glucagon administration to study the effect on the LMW iron pool.

A 31P NMR study of preconditioned isolated perfused rat heart exposed to intermittent ischemia

Exposure to a short ischemic period (ischemic preconditioning, IP) will protect the heart from damage following a subsequent longer ischemic episode. The aim of the study was to test whether IP is cardioprotective in the setting of repeated ischemia-reperfusion cycles. Thus, Langendorff-perfused hearts, exposed to IP, were subjected to three consecutive ischemia-reperfusion (10/15 min) cycles. Myocardial energetics, manifested by 31P NMR spectroscopy, was correlated with hemodynamics. ATP recovery was significantly higher for the IP group compared with control (P < 0.02) during reperfusions. However, there was no significant difference in ATP recovery during the three ischemic intervals. The supernormal recovery of phosphocreatine recorded during reperfusion was lower for the IP group (approximately 120%) compared with control (approximately 135%, P < 0.065). Better recovery of the left ventricular-developed pressure was noted during reperfusions for the IP group and became significant only during the last reperfusion (86% versus 68%, P < 0.025). In conclusion, the above results support prolonged IP cardioprotection.

The role of substance P in myocardial dysfunction during ischemia and reperfusion.

Impairment of myocardial contraction ("myocardial stunning") occurs during reperfusion after short ischemic periods. Substance P (SP) is widely distributed in heart and can be released by various stimuli including myocardial hypoxia. Our previous study shows SP has a negative inotropic effect in guinea pig heart. The objective of this study was to investigate whether SP contributes to the myocardial stunning after brief global ischemia. Guinea pig hearts in a Langendorff preparation were subjected to 15 min of global ischemia followed by 60 min reperfusion. Experiments were performed without and with pretreatment with neurokinin-1 (NK1) receptor antagonists, spantide (10(-6)M) or CP-99,994-01 (10(-6)M) in order to study the role of SP. Experiments were also performed in hearts which were perfused with atropine, phentolamine, and nadolol (10(-6)M each) to examine the role of neurotransmitters and autonomic receptors. A group of hearts obtained from capsaicin-pretreated guinea pigs was also investigated. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), heart rate, and perfusion pressure were monitored. At the end of reperfusion, the LVDP of control hearts recovered to only 55 +/- 6% (+/- SEM) of preischemic baseline and the LVEDP increased significantly (P > 0.05). With pretreatment with spantide or CP-99,994-01, LVDP recovered to 88 +/- 2% or 78 +/- 2% of the preischemic baseline, respectively. The LVEDP of these hearts was not different from preischemic baseline and much smaller than in control hearts. There were no differences in heart rate and perfusion pressure compared to baseline among all groups. Similar results were obtained in hearts perfused with autonomic blockers. However, recoveries of LVDP and LVEDP were faster in hearts perfused with autonomic blockers during the first 10 min of reperfusion. Pretreatment with capsaicin also significantly improved recovery of LVDP and LVEDP. In conclusion, substance P is involved in postischemic myocardial dysfunction and neurokinin-1 receptors mediate this action. The NK1 receptor antagonists may be useful in prevention of "myocardial stunning".

Ischemic Preconditioning—A New Concept in Orthopedic and Reconstructive Surgery

The duration of tourniquet-induced ischemia during orthopedic and reconstructive surgery is limited by the risk of ischemia and reperfusion injury to skeletal muscle. This study evaluated the potential of ischemic preconditioning (short periods of ischemia with intermittent reperfusion) to improve skeletal muscle function after ischemia and reperfusion in a rodent model. Preconditioning was found to improve force, contractility, and performance and to decrease fatigue of skeletal muscle. In contrast, energy-rich phosphates, measured concurrently, were not affected by preconditioning, suggesting mechanisms other than energy preservation to be involved. In summary, preconditioning may enable prolongation of orthopedic and reconstructive procedures.

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long‐Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD‐GP180

The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M(r) 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M(r) 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro "ischemia" was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N2 instead of O2 and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca2+ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca2+ was omitted from the incubation media.(ABSTRACT TRUNCATED AT 250 WORDS)

Bimakalim, an ATP-sensitive potassium channel opener, mimics the effects of ischemic preconditioning to reduce infarct size, adenosine release, and neutrophil function in dogs.

The primary goal of the present study was to determine whether the infarct size-reducing effect of preconditioning is associated with an increase in adenosine release from the ischemic myocardium during a prolonged occlusion period or the subsequent reperfusion period and by a decrease in neutrophil infiltration. A second objective was to determine whether bimakalim, a KATP channel opener, mimics the effects of ischemic preconditioning. Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 3 hours of reperfusion. In the preconditioning group, 5 minutes of LAD occlusion followed by 10 minutes of reperfusion was elicited before the 60-minute occlusion period. In two other groups, bimakalim 1 microgram/kg bolus followed by a 0.05 micrograms.kg-1.min-1 infusion or an equivalent volume of saline was administered intravenously 15 minutes before occlusion and continued until the time of reperfusion. In a final group, bimakalim was administered 10 minutes before reperfusion and continued until the end of the experiment. To measure the release of adenosine from the ischemic region, coronary venous blood samples were collected at various times during ischemia and after reperfusion, and the concentration of adenosine was measured. Myocardial infarct size was determined by triphenyl tetrazolium chloride; transmural myocardial blood flow, by radioactive microspheres. Transmural myeloperoxidase (MPO) activity, an index of neutrophil infiltration in the area at risk, was also measured. Preconditioning produced a marked reduction in infarct size (9.8 +/- 3.0% versus 28.6 +/- 5.2% in the control group, mean +/- SEM); adenosine release at 5, 10, 15, and 30 minutes of the 3-hour reperfusion period; and transmural MPO activity in the risk area. Similarly, pretreatment with bimakalim resulted in reductions in infarct size, adenosine release, and transmural MPO activity to an extent almost identical to that of preconditioning. When bimakalim was administered 10 minutes before reperfusion, the drug also produced a significant reduction in infarct size and transmural MPO activity; however, no significant reduction in coronary venous adenosine concentrations was observed. There were no significant differences in collateral blood flow between groups. These results indicate that myocardial preconditioning in the canine heart produced by a short period of ischemia or a KATP channel opener is not mediated by an increase in adenosine release, as measured by coronary venous adenosine concentrations, during 60 minutes of occlusion or the initial 30 minutes of reperfusion. A significant reduction in transmural MPO activity in the ischemic area also appears to result from KATP channel activation and may play a role, at least in part, in the reduction in infarct size observed, particularly when a KATP channel opener is administered just before reperfusion.

Trimetazidine effects on the damage to mitochondrial functions caused by ischemia and reperfusion

Trimetazidine (TMZ) is an anti-ischemic compound whose precise mode of action is unknown, although several studies have suggested a metabolic effect, and there have been reports of protection of mitochondria against oxidative stress damage. Using a Langendorff rat heart model, we examined the effects of TMZ on the mitochondrial damage following 30 minutes of ischemia and 5 minutes of reperfusion. Mitochondrial respiration with succinate, glutamate-malate and ascorbate-N,N,N′,N′-tetramethylphenylenediamine (TMPD) as substrates was significantly decreased following ischemia-reperfusion. Preperfusion with 10 −5 M TMZ had no effect on these rates in normoxic or ischemic hearts. However, 10 −3 M TMZ significantly decreased the glutamate-malate rate in mitochondria from normoxic hearts, and this rate was not further decreased following ischemia-reperfusion, and 10 −3 M TMZ also partially protected ascorbate-TMPD activity. The effect on glutamate-malate was probably due to an inhibition of complex I by TMZ, which specifically inhibited reduced nicotinamide-adenine-dinucleotide-cytochrome c reductase and complex I in lysed mitochondria. We also studied the effects of TMZ on the activity of pyruvate dehydrogenase (PDH) in normoxic and ischemic hearts perfused with 0.5 m M palmitate, which caused the enzyme to be almost completely inactivated. After short periods of ischemia (10–20 minutes) the PDH inactivation by palmitate was progressively lost. Preperfusion with 10 −5 M TMZ had a tendency to decrease lactate dehydrogenase release, accompanied by a maintenance of the inhibition of PDH by palmitate. This may allow the heart to oxidize fatty acids preferentially during reperfusion, hence removing possible toxic acyl esters.

Trimetazidine effects on the damage to mitochondrial functions caused by ischemia and reperfusion

Trimetazidine (TMZ) is an anti-ischemic compound whose precise mode of action is unknown, although several studies have suggested a metabolic effect, and there have been reports of protection of mitochondria against oxidative stress damage. Using a Langendorff rat heart model, we examined the effects of TMZ on the mitochondrial damage following 30 minutes of ischemia and 5 minutes of reperfusion. Mitochondrial respiration with succinate, glutamate-malate and ascorbate-N,N,N',N'-tetramethylphenylenediamine (TMPD) as substrates was significantly decreased following ischemia-reperfusion. Preperfusion with 10(-5) M TMZ had no effect on these rates in normoxic or ischemic hearts. However, 10(-3) M TMZ significantly decreased the glutamate-malate rate in mitochondria from normoxic hearts, and this rate was not further decreased following ischemia-reperfusion, and 10(-3) M TMZ also partially protected ascorbate-TMPD activity. The effect on glutamate-malate was probably due to an inhibition of complex I by TMZ, which specifically inhibited reduced nicotinamide-adenine-dinucleotide-cytochrome c reductase and complex I in lysed mitochondria. We also studied the effects of TMZ on the activity of pyruvate dehydrogenase (PDH) in normoxic and ischemic hearts perfused with 0.5 mM palmitate, which caused the enzyme to be almost completely inactivated. After short periods of ischemia (10-20 minutes) the PDH inactivation by palmitate was progressively lost. Preperfusion with 10(-5) M TMZ had a tendency to decrease lactate dehydrogenase release, accompanied by a maintenance of the inhibition of PDH by palmitate. This may allow the heart to oxidize fatty acids preferentially during reperfusion, hence removing possible toxic acyl esters.

Adenosine and PAF dependent mechanisms lead to myocardial reperfusion injury by neutrophils after brief ischaemia

The aim was to establish whether polymorphonuclear neutrophils can, by themselves, elicit depression of postischaemic heart function immediately after short periods of ischaemia, and to examine the involvement of endogenous adenosine and platelet activating factor (PAF) in the observed phenomena. Isolated buffer perfused guinea pig hearts performing pressure-volume work under standardised conditions were subjected to 15 min of global ischaemia. Constant flow reperfusion (5 ml.min-1) was carried out in the presence or absence of homologous neutrophils (approximately 2000 cells.microliters-1 perfusate). After 15 min of reflow, work was resumed and functional recovery assessed another 20 min later. In hearts perfused only with Krebs-Henseleit buffer, postischaemic heart function recovered to 67(SEM 3)% (n = 13) of the preischaemic value. As early as the first minute of reperfusion, the application of neutrophils already led to a significant decrease in recovery to 39(3)% (n = 12; P < 0.05). Without ischaemia, neutrophils did not have any deleterious effect, recovery of external heart work amounting to 91(4)% (n = 6). Adenosine concentrations measured in the coronary effluent after ischaemia were substantially increased during reperfusion from preischaemic values of < 20 nM to about 800 nM. The adenosine A1 receptor antagonist dipropyl-8-cyclopentyl-xanthine prevented the neutrophil dependent loss of heart function, as did application of the PAF receptor antagonist WEB 2086, recoveries being 60(4)% (n = 6) and 58(8)% (n = 6), respectively. In contrast, the A2 antagonist 3,7-dimethyl-1-propargyl-xanthine tended to worsen the neutrophil induced dysfunction of the heart, recovery amounting to 18(2)% (n = 5). Even after brief ischaemia, neutrophils introduced into the coronary system can exacerbate reperfusion injury. Adenosine, through its A1 receptor, and PAF appear to play a significant role as mediators of this action.