Normothermic Ischemia Research Articles

Effects of hyperbaric oxygen on the replanted extremity subjected to prolonged warm ischaemia

In this investigation, the influence of hyperbaric oxygen (HBO) therapy on the survival of a replanted extremity subjected to prolonged warm ischaemia is evaluated. Among the relative contraindications to replantation are prolonged warm ischaemia time, since an obstruction to blood reflow ('no-reflow phenomenon') may occur in amputated parts that are subjected to more than 6h of warm ischaemia. Twenty-three rat hindlimbs were amputated and subjected to 4h of normothermic ischaemia. The average weight of the animals was 500 gm, and re-plantation of the hindlimb was performed by bone fixation followed by microvascular anastomosis of the femoral vessels. Limb re-vascularisation was confirmed at the end of all procedures by the milk test, clinical assessment and pulse oximetry recordings (>90%). Eleven animals served as a control group and no further therapy was instituted, whereas 12 animals served as the study (replantation) group and were subjected to HBO therapy for 3 days postoperatively. The therapy was conducted in a large animal chamber for 90 min at 2.5 ata. Limb survival was assessed by capillary refill upon compression, skin turgor assessment and colour. Confirmation of clinical findings was conducted with daily pulse oximetry readings of >90%. Animals were followed up for 7 days at which point all animals were euthanized or were euthanized earlier if a non-viable limb was present. Two of the 11 limbs in the control group survived following re-plantation, whereas eight of the 12 limbs in the experimental HBO group survived at least 7 days following replantation. This difference was statistically significant (p=0.0361) using chi-square analysis and Fisher's exact test. Although re-plantation of an amputated extremity after prolonged warm ischaemia is considered a relative contraindication due to the possibility of poor outcomes, our results indicate that the window for replantation may be increased if adjunctive HBO therapy is employed in the postoperative period.

Ischemia-reperfusion injury activates early extracellular matrix processing and expression of endostatin in the heart with differential effects of temperature

Acute ischemia is a well-known inductor of extracellular matrix (ECM) remodeling, which leads to the development of congestive heart failure and is associated with left ventricular dilatation. Here we investigate the timecourse of ECM processing with release of endostatin (ES) and other low-molecular-weight fragments during early ischemia-reperfusion of the heart. In this blinded study, 30 pigs were randomized to 60 min of global myocardial ischemia at either 4 or 37 degrees C or served as control. Five transmyocardial tissue samples were collected at baseline and after ischemia within 150 min of reperfusion. Collagen XVIII cleavage products of 10-75 kDa including ES (25 kDa) were analyzed using the Western blot and ELISA method, and creatin kinase as marker of myocardial injury was determined in samples collected from the coronary sinus. We demonstrate that processing of the extracellular matrix protein collagen XVIII starts during early reperfusion, as we observed a significantly increased expression of cleavage products at 10 and 75 kDa as well as ES at 150 min of normothermic ischemia-reperfusion. We further demonstrate a differential processing of collagen XVIII depending on temperature conditions during myocardial ischemia, as an increase in cleavage products was observed after normothermic ischemia only; however, expression of ES and other fragments remained unchanged after hypothermic ischemia-reperfusion and in controls. In conclusion, this blinded study first demonstrated that processing of extracellular matrix started early after ischemia-reperfusion and depends on temperature conditions. These findings may contribute to a broader understanding of matrix processing after ischemia-reperfusion.

Conditioning of myocutaneous flaps

The improvement of the ischaemic tolerance of myocutaneous flaps is of clinical importance and hence the subject of numerous investigations. In an attempt to increase the ischaemic tolerance, 20 myocutaneous flaps (rectus abdominis muscle) in pigs were elevated and perfused with various, established solutions prior to the onset of ischaemia. The flaps were elevated, utilizing the superior epigastric vessels as the pedicle. Ten flaps were flushed with the University of Wisconsin solution, five with the Euro-Collins solution and the last five with a Ringer-Lactate solution, prior to the 6h long, normothermic ischaemia. On the day of operation, the first, third, fifth, seventh and tenth postoperative day clinical examinations and thermography were performed as well as biopsies. Additionally, on the tenth postoperative day, the rate of necrosis was determined morphometrically as the average of three measurements. Ten days after surgery, the flaps pretreated with the University of Wisconsin solution displayed a vital surface area of 89%, the Euro-Collins solution 23% and the Ringer-Lactate solution 14%. Histologically, muscle tissue proved to be more susceptible to ischaemia than skin. Regarding the rectus abdominis flap in a pig model, the University of Wisconsin solution proved superior in the prevention of ischaemic injury compared with the Euro-Collins solution and Ringer Lactate. In accordance with the literature, muscle tissue proved to be more susceptible to ischaemia than skin in our study.

Fundamental Efforts toward the Development of a Therapeutic Cocktail with a Manifold Ameliorative Effect on Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury is mediated by various mechanisms. The present study was designed to evaluate the effect of a multiple pharmacological approach toward ischemia/reperfusion injury reduction. The left liver lobe of Sprague-Dawley rats underwent normothermic ischemia for 90 minutes after a cocktail (glycine, taurine, alanine, arginine, and prednisolon) intravenous administration. Controls received normal saline. Liver injury (transaminases, histology) and cellular activation [Kupffer cell phagocytosis, production of tumor necrosis factor-alpha (TNFalpha), and prostaglandin E-2 (PGE(2))], as well as microcirculation and leukocyte-endothelial interaction (in vivo microscopy), were assessed. Whereas in controls a substantial increase of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase to 2,341+/-477, 1,442+/-262, and 1,973+/-73 U/L, respectively, was measured eight hours after reperfusion, the cocktail significantly reduced the increase of enzymes by 33-80% (P<0.05). Further, necrosis, index of liver damage, and index of leukocyte infiltration significantly decreased after the cocktail (P<0.05). Moreover, the cocktail improved acinar and sinusoidal perfusion, while the sinusoidal diameters, leukocyte-endothelial interaction, Kupffer cell phagocytic activity, and TNFalpha/PGE(2) serum levels were significantly reduced, the latter by 86% and to 1.64-fold, respectively. This study depicts that multifaceted pharmacological tackling of ischemia/reperfusion injury is feasible and protects rat liver tissue from warm ischemia/reperfusion injury.

Abstract 2316: Ranolazine, a Late Sodium Current Inhibitor, as an Adjunct to Cardioplegia Further Reduces Contracture during Ischemia and Reperfusion

Although cardioplegia (CP) protects myocardium during ischemic cardiac arrest, cardioplegic preservation can be suboptimal for increasingly complex cardiac surgical patients. Ranolazine (Ran), a novel antianginal agent, was recently shown to mitigate ischemia-induced Ca 2+ overload via its inhibitory effects on the late Na + current, but the benefit of Ran during cardioplegic arrest is not known. The purpose of the study was to investigate the therapeutic potential of Ran as an adjunct to CP. Hearts isolated from female Sprague-Dawley rats were Langendorff-perfused and exposed to normothermic global ischemia for 40 min followed by 30-min reperfusion (I/R). Three groups were studied: control buffer (n = 10); crystalloid CP (Fremes, n = 12); CP supplemented with Ran (n = 12). CP was bolus-injected with or without Ran at the time of ischemia. End-diastolic pressure (EDP), developed pressure, dP/dt min , and dP/dt max were measured throughout the study. CP significantly delayed onset of ischemic contracture (defined as a time to 20 mmHg of EDP), compared to control (25 ± 2 min in CP alone vs. 12 ± 1 min in control, p &lt; 0.05). Ran added to CP further delayed the time to contracture (34 ± 2 min in Ran, p &lt; 0.05 compared to CP alone). Consistent with these findings, the area under the diastolic pressure curve during the entire period of ischemia was significantly smaller in CP + Ran versus CP alone (p &lt; 0.05). I/R caused dramatic elevations in EDP during reperfusion. CP lessened the extent of the reperfusion contracture (32 ± 3 mmHg in CP alone vs. 76 ± 3 mmHg in control, p &lt; 0.05). Addition of Ran to CP further lessened the contracture (17 ± 2 mmHg in Ran, p &lt; 0.05 compared to CP alone). I/R severely depressed systolic cardiac contractile function. The impairment in contractile function was significantly reduced by CP, as well as CP + Ran, but there was no statistical difference between the two groups. While CP alone reduced the onset of cardiac contracture in this model of I/R, addition of Ran to CP further enhanced the cardioprotection. These results suggest the potential therapeutic efficacy of Ran as an adjunct to CP and further support the protective role of late Na + current inhibition.

Cardioprotection by Ouabain and Digoxin in Perfused Rat Hearts

This work was aimed at determining the cardioprotective effect of digitalis glycosides in rat heart, and to relate it with Na, K-ATPase inhibition and ERK1/2 activation. Isolated working rat hearts were perfused in the presence of ouabain or digoxin, which were used at concentrations ranging from 10 to 10 M. The hearts were then subjected to 30 minutes of global normothermic ischemia followed by 120 minutes of retrograde reperfusion; irreversible tissue injury was determined on the basis of triphenyltetrazolium chloride staining. Significant cardioprotection was observed with 10 M and 10 M ouabain (ischemic injury averaged 7.0 +/- 3.5% and 8.3 +/- 0.6% versus 37.3 +/- 2.0% in controls, P < 0.01 in each case). Hearts treated with digoxin showed decreased ischemic injury at 10 M and 10 M (18.0 +/- 1.5% and 14.2 +/- 1.0%, P < 0.01 versus control in both cases). In parallel experiments, ERK2 phosphorylation was increased by 10 to 10 M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10 to 10 M digoxin. The cardioprotective effect was not related to Na, K-ATPase inhibition, since Rbuptake was not significantly different between control and treated hearts.

Diazepam delays the death of hippocampal CA1 neurons following global ischemia

Although diazepam provides limited long term neuroprotection, it may be useful for expanding the therapeutic time window after stroke by delaying neuronal death. However, it is not known to what extent diazepam maintains normal cellular structure and function in the first few days after ischemia. We used histological, immunohistochemical and electrophysiological endpoints to address this question. Gerbils underwent 5 min of global ischemia followed by 10 mg/kg diazepam (D) given 30 and 90 min later. Other animals were subjected to sham surgery, normothermic ischemia (I) or ischemia at 32 °C (Hypo). Postischemic brain temperature was regulated at ∼ 37 °C for 24 h. Gerbils in the D and I groups were sacrificed 1, 2 and 3 days after ischemia. Sham and Hypo gerbils were sacrificed on day 3. CA1 cell counts, MAP2 staining and CA1 field potentials were performed at each survival time. Hypothermia prevented CA1 necrosis, preserved MAP2 integrity and maintained CA1 field potential amplitude. Ischemic gerbils showed a significant reduction in these 3 outcome measures by day 3. Diazepam-treated gerbils exhibited near normal levels of CA1 neurons and MAP2 staining. Most importantly, CA1 field potentials were similar to sham values and significantly preserved relative to non-treated ischemic gerbils. Diazepam maintains near normal structural and functional integrity up to 3 days after a global ischemic insult. As such, this drug may be useful for extending the therapeutic time window after cardiac arrest, stroke and related disorders.

Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver

Phosphoregulation of signal transduction pathways is a complex series of reactions that may modulate the cellular response to ischemia-reperfusion (I-R). The aim of this study was to evaluate the effect of normothermic liver I/R-induced apoptosis on phosphorylation and activation of signal proteins in tyrosine kinase pathways. In rats, a segmental normothermic ischemia of the liver was induced for 120 min. Liver apoptosis was determined using terminal deoxynucleotide-transferase-mediated deoxyuridine triphosphate nick end labeling assay, and activity of caspases-3 and -7 was determined by fluorescence. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis. Normothermic I-R resulted in increased in vivo caspases-3 and -7 activity and in liver apoptosis. Shc tyrosine phosphorylation and activation of ERK1/2 were increased after reperfusion, while tyrosine phosphorylation of IRS-1 and activation of PKB/Akt were decreased. Normothermic liver I-R leads to increased apoptosis and to modifications in protein tyrosine phosphorylation pathways.

Liver Apoptosis Following Normothermic Ischemia-Reperfusion: In Vivo Evaluation of Caspase Activity by FLIVO Assay in Rats

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular apoptosis. Caspase activation is involved in the initiation and execution of apoptosis. The aim of this study was to determine in vivo caspase activity in normothermic liver I-R in rats. Segmental normothermic ischemia of the liver was induced for 120 minutes in rats. After intravenous injection of the green probe FLIVO ™, in vivo caspase-3- and -7-specific activity was determined using fluorescence microscopy, in either nonischemic or ischemic liver lobes at 3 and 6 hours after reperfusion. Liver apoptosis was assessed by the deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Fluorescence microscopy showed that in vivo caspase-3- and -7-specific activities were significantly increased ( P < .005) in ischemic lobes at 3 and 6 hours of reperfusion, compared with nonischemic liver lobes. Quantitative analysis of apoptotic cells measured by the TUNEL method showed a significant increase among apoptotic cells in ischemic lobes at 3 and 6 hours after reperfusion ( P < .005), compared with nonischemic liver lobes. In conclusion, 120-minute normothermic liver I-R resulted in increased caspase-3- and -7-specific activities and in liver cell apoptosis.

Liver HIF-1 Alpha Induction Precedes Apoptosis Following Normothermic Ischemia-Reperfusion in Rats

Apoptosis plays an important role in ischemia-reperfusion (I-R) injury during liver transplantation. The hypoxia-inducible factor alpha (HIF-1α) may trigger liver apoptosis following I-R through the induction of hypoxically regulated genes. The aim of this study was to evaluate the effect of normothermic liver I-R on HIF-1α expression and apoptosis in rats. Segmental normothermic ischemia of the liver was induced in rats for 120 minutes. Liver extracts from either ischemic or nonischemic lobes were prepared at 0, 1, 3, and 6 hours after reperfusion. Liver HIF-1α protein expression was examined by Western blot analysis. Liver apoptosis was quantified using terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling assay. Normothermic I-R resulted in a significant ( P < .05) increase in liver HIF-1α protein levels 1 and 3 hours after reperfusion. Liver apoptosis was significantly ( P < .005) increased at 3 and 6 hours after reperfusion. In conclusion, normothermic liver I-R leads to increased liver expression of HIF-1α and apoptosis.

Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation

Inhibition of mitochondrial permeability transition pore (MPTP) opening at reperfusion is critical for cardioprotection by ischemic preconditioning (IP). Some studies have implicated mitochondrial protein phosphorylation in this effect. Here we confirm that mitochondria rapidly isolated from preischemic control and IP hearts show no significant difference in calcium-mediated MPTP opening, whereas IP inhibits MPTP opening in mitochondria isolated from IP hearts following 30 minutes of global normothermic ischemia or 3 minutes of reperfusion. Analysis of protein phosphorylation in density-gradient purified mitochondria was performed using both 2D and 1D electrophoresis, with detection of phosphoproteins using Pro-Q Diamond or phospho-amino-specific antibodies. Several phosphoproteins were detected, including voltage-dependent anion channels isoforms 1 and 2, but none showed significant IP-mediated changes either before ischemia or during ischemia and reperfusion, and neither Western blotting nor 2D fluorescence difference gel electrophoresis detected translocation of protein kinase C (alpha, epsilon, or delta isoforms), glycogen synthase kinase 3beta, or Akt to the mitochondria following IP. In freeze-clamped hearts, changes in phosphorylation of GSK3beta, Akt, and AMP-activated protein kinase were detected following ischemia and reperfusion but no IP-mediated changes correlated with MPTP inhibition or cardioprotection. However, measurement of mitochondrial protein carbonylation, a surrogate marker for oxidative stress, suggested that a reduction in mitochondrial oxidative stress at the end of ischemia and during reperfusion may account for IP-mediated inhibition of MPTP. The signaling pathways mediating this effect and maintaining it during reperfusion are discussed.

Moderate hypothermia as a rescue therapy against intestinal ischemia and reperfusion injury in the rat*

Moderate hypothermia is protective when applied throughout experimental intestinal ischemia and reperfusion (I/R). However, therapeutic intervention is usually possible only after ischemia has occurred. The aim of this study was to evaluate moderate hypothermia when applied at reperfusion as a rescue therapy for intestinal I/R. Prospective, randomized, controlled experiment. University research laboratory. Adult male Sprague-Dawley rats (240-300 g). In experiment I, rats underwent 60 mins of normothermic intestinal ischemia (36-38 degrees C) plus 300 mins of reperfusion at either normothermia or moderate hypothermia (30-32 degrees C) with or without rewarming. Hemodynamics were measured invasively and survival was assessed. In experiment II, rats underwent 60 mins of normothermic ischemia plus 120 mins of reperfusion at either normothermia or moderate hypothermia. At kill, organs and a blood sample were collected. In experiment I, all normothermic I/R rats died within 197 mins of reperfusion after developing severe tachycardia and hypotension, whereas hypothermic rats, with or without rewarming, were alive at 300 mins of reperfusion (p < .001 vs. I/R normothermia) and were hemodynamically stable. In experiment II, normothermic reperfusion caused histologic and biochemical damage to the gut, hepatic energy failure, and inflammatory infiltration of the lung. However, hypothermia reduced injury to the reperfused ileum and prevented distant organ injury by counteracting energy failure in the liver, systemic overproduction of nitric oxide, altered cardiac fatty acid metabolism, and infiltration of inflammatory cells in the lungs. Hypothermia applied as a rescue therapy for intestinal I/R abolishes mortality even after rewarming. Hypothermic protection during early reperfusion appears to be mediated by several pathways, including prevention of intestinal and pulmonary neutrophil infiltration, reduction of oxidative stress in the ileum, and preservation of cardiac and hepatic energy metabolism. Moderate hypothermia may improve outcome in clinical conditions associated with intestinal I/R.

Cardioprotection from ischaemia–reperfusion injury: Dissecting the PI3K/AKT pathway with chemical inhibition of PTEN

Stimulation of the prosurvival PI3K/AKT pathway, as triggered in ischaemic preconditioning (IPC), protects the heart from ischaemia–reperfusion injury (IRI). The main endogenous negative regulator of this pathway is phosphatase and tensin homologue deleted on chromosome 10 (PTEN). We hypothesised that PTEN inhibition will confer myocardial protection to IRI. Isolated C57BL/J6 mouse hearts in a Langendorff constant pressure system were subjected to 35 min global normothermic ischaemia and 30 min reperfusion.

Lack of the effect of superoxide dismutase and catalase on Na+,K+-ATPase activity in stunned rabbit hearts

Reactive oxygen species (ROS) have been implicated in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. In this study, we examined protective effects of antioxidant enzymes, superoxide dismutase (SOD) and catalase, against ischemia/reperfusion-induced cardiac dysfunction and inhibition of Na+,K+-ATPase activity. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min of global normothermic ischemia followed by 10 min reperfusion. The hearts treated with SOD plus catalase did not show significant recovery of left ventricular (LV) end-diastolic pressure compared with untreated ischemic reperfused hearts. Treatment with antioxidants had no protective effects on developed LV pressure or its maximal positive and negative first derivatives (+/-LVdP/dt). Myocardial stunning was accompanied by significant loss in sarcolemmal Na+,K+-ATPase activity and thiol group content. Inhibition of enzyme activity and oxidation of SH groups were not prevented by antioxidant enzymes. These results suggest that administration of SOD and catalase in perfusate do not protect significantly against cardiac dysfunction in stunned rabbit myocardium.

ISCHEMIC AND PEROXYNITRITE PRECONDITIONING EFFECTS IN CHRONIC HYPOXIC RAT LUNG

Chronic hypoxic pulmonary hypertension is characterized by vasoconstriction and vascular remodeling and impaired endothelial nitric oxide (NO) production. Although ischemic preconditioning of the lung leads to protective effect against ischemic reperfusion injury, the mechanisms of this protection are not well documented in the lung. The aim of this study was to investigate the effects of chronic hypoxia on preconditioning induced by ischemia or peroxynitrite in isolated rat lungs. The isolated rat lung, from exposed to hypobaric hypoxia for 21 days, was mounted on a modified Langendorff perfusion apparatus. Lungs were preconditioned by either 5 minutes' ischemia and 5 minutes' reperfusion or 10 μ M peroxynitrite prior to 2 hours of normothermic ischemia. Although ischemia-reperfusion or peroxynitrite preconditioning markedly reduced KCl responses on perfusion pressure, phenylephrine-induced responses were not significantly modified. Pretreatment of the hypoxic lungs with peroxynitrite scavenger, uric acid, or poly (ADP-ribose) synthase inhibitors (PARS), 3-aminobenzamide (3-AB) or nicotinamide, did not modify the KCl- and phenylephrine-induced responses in chronic hypoxic lungs. There were also no marked differences either in wet to dry weight ratio or malondialdehyde levels of chronic hypoxic lungs. These results imply that preconditioning does not occur in the chronic hypoxic rat lungs.

Cyclosporine action on kidneys of rats submitted to normothermic ischaemia and reperfusion

To verify if rat kidneys lesioned by ischaemia followed by reperfusion are affected by cyclosporine A (CsA). Male Wistar rats were randomly divided into three groups, control (GS) and experimental (G1 and G2). G1 was subdivided in two: G1A composed of animals submitted to 60 minutes ischaemia and G1C with the same ischaemic procedure associated to 20 mg/kg/day CsA. Group G2 was subdivided and treated in the same way as G1 except that ischaemia was applied only for 40 minutes. Clamping the left renal artery followed by right side nephrectomy induced kidney ischaemia. Serum urea and creatinine were quantified on the day of surgery (D0) and in the following day (D1). Twenty four hours after reperfusion the left kidney was removed and histologically analyzed. Group GS had normal values for urea and creatinine both on D0 and D1 and did not show structural alterations. Renal function was not significantly different when G2C was compared to GS (p>0.05). Tissue lesions were smaller in G2C than in the other groups. Renal function was protected by CsA, which also reduced tissue lesions in the kidneys of rats submitted to 40 minutes ischaemia.

Cardioprotection of neonatal heart using normothermic hyperkalaemia: the importance of delivery and terminal cardioplegia

Cardioprotection of immature hearts remains controversial and largely based on the use of hypothermic cardioplegia. Recent clinical trials in pediatric open-heart surgery suggest that normothermic cardioplegic arrest is also cardioprotective. However, the advantages of using normothermic cardioplegia delivered as single- or multi-dose with or without terminal cardioplegia are unknown. This work investigates the efficacy of these techniques and the mechanism(s) underlying their protective effect. Neonatal (7-10 days) rabbit hearts in a working mode were exposed to normothermic global ischemia (60 or 90 min) protected with one of the following cardioplegic (hyperkalaemic buffer) protocols: single-dose, multi-dose infused every 30 min, single-dose or multi-dose with terminal cardioplegia. The extent of functional recovery (e.g., aortic and coronary flow), ischemic stress (e.g., myocardial ATP, lactate) and reperfusion injury (lactate dehydrogenase (LDH) release) were assessed. Recovery following 60 min global ischemia was improved (p < 0.05) by single-dose and multi-dose cardioplegic delivery (from 5% to 60% and 80%, respectively). Improved recovery was augmented by 2 min terminal cardioplegia (to 90% and 97% for single-dose and multi-dose, respectively). Extending ischemia to 90 min with single-dose resulted in 0% recovery that was not improved by 2 min terminal cardioplegia. However, 5 min (not 10 min) terminal cardioplegia significantly improved recovery (32%). Multi-dose followed by 5 min terminal cardioplegia resulted in full recovery. Cardioprotective interventions were associated with a reduction in LDH release and attenuated changes in myocardial metabolites. During normothermic cardioplegic arrest of neonatal heart: (i) multi-dose is superior to single-dose; (ii) terminal cardioplegia confers additional protection to single-dose and multi-dose; and (iii) protection is likely to be due to metabolic preservation.

Effects of various protocols of ischemic preconditioning on rat tram flaps

We used rat transverse rectus abdominis musculocutaneous (TRAM) flaps to investigate whether the ischemic cycle duration and number of cycles influenced the effectiveness of ischemic preconditioning (IPC). First, the animals were divided into one control group and four treatment groups. Controls received no preconditioning; flaps with IPC were treated by two cycles of pedicel clamping and reperfusion for either 5 or 10 min. Then the best of IPC protocols in first part was selected, and flaps were subjected to one or three cycles of IPC before 4 h of normothermic ischemia. The preconditioned flap survival area had improved 2.5-3 times over that of the control area by postoperative day 7. Serum creatine phosphokinase (CPK), water content, and malondialdehyde (MDA) in muscle were significantly decreased in the preconditioned groups after 4 h of reperfusion. Especially, 2 and 3 cycles of the 10/5-min combination were more effective than other protocols of IPC.

Preconditioning With Na+/H+ Exchange Inhibitor HOE642 Reduces Calcium Overload and Exhibits Marked Protection on Immature Rabbit Hearts

Inhibition of Na/H exchanger isoform-1 (NHE1) has shown significant protection in adult myocardium during ischemia/reperfusion injury; however, the effect is unclear in immature myocardium. We evaluated the effects of HOE642 (a potent, highly selective NHE1 inhibitor) preconditioning on immature rabbit hearts. Twenty immature (2-3 weeks old) New Zealand white rabbits were randomly divided into the control group (n = 10) and the HOE642 preconditioning group (n = 10). The immature isolated hearts were subjected to 45 minutes of normothermic global ischemia plus 60 minutes of reperfusion after being established on the Langendorff apparatus. During reperfusion, the recovery rates of cardiac function (LVDP, +dp/dtmax, -dp/dtmax, and coronary flow) were about 90% in the HOE642 treated group and about 50% in the control group (p < 0.05). HOE642 preconditioning can significantly decrease the release of cardiac specific enzymes CK, CK-MB and LDH (p < 0.05) and the myocardial water content (p < 0.05). Meanwhile, HOE642 markedly attenuated intracellular calcium overload (265.8 +/- 41.1 vs. 500.7 +/- 60.8 mg/kg dry wt) (p < 0.01). The blinded ultrastructural assessment under transmission electron microscopy illustrated that preconditioning with HOE642 produced evident myocyte salvage. This study demonstrates that preconditioning with HOE642 provides a significant protection during ischemia/reperfusion injury in immature myocardium, mostly by reducing myocardial calcium overload.

Protection of cellular and mitochondrial functions against liver ischemia by N-benzyl- N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine (BHDP), a sigma 1 ligand

We investigated the antiischemic properties of a new compound N-benzyl- N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine (BHDP), having high affinity and selectivity for the sigma 1 receptor, in two different models of ischemia. The first was an experimental model of rat liver normothermic ischemia-reperfusion. Rats were pretreated with different doses of BHDP (0.5, 2.5 or 10 mg/kg/day, or solvent alone) and subjected to 90 min normothermic ischemia followed by either 30 or 120 min reperfusion. The second model was a hypothermic model of ischemia in which livers were incubated for 24 h at 4 °C in a preservation solution in the absence or presence of increasing BHDP concentrations (0.5, 2.5 or 10 μg/ml). These different ischemic conditions induced huge alterations in hepatocyte functions (membrane leakage of alanine aminotransferase and aspartate aminotransferase, decreased metabolic capacities evaluated by the ability of the liver to transform lidocaine, alterations of mitochondrial functions characterized by a decrease in ATP synthesis and the appearance of histological damages). Pretreatment of rats with BHDP alleviated these deleterious ischemia-reperfusion effects in a dose-dependent manner at both the cellular and mitochondrial levels. The protection of mitochondrial functions was almost complete at a dosage of 10 mg/kg/day during normothermic ischemia and 10 μg/ml in the preservation liquid during hypothermic ischemia. In addition, BHDP significantly reduced the histological damage. These data demonstrate that BHDP protects liver against the deleterious effects of ischemia-reperfusion and suggest that sigma 1 receptors play an important role in the protective effect.