Early Phase Of Reperfusion Research Articles

Abstract 399: Reactive Oxygen Species in Myocardial Ischemic Injury and Protection

Myocardial injury following ischemia/reperfusion (I/R) is a common clinical scenario in patients suffering from ischemic heart disease. An excessive production of reactive oxygen species (ROS) during the early phase of reperfusion following myocardial ischemia has been proposed to contribute to reperfusion injury. Paradoxically, the ROS has also been recognized as a trigger of pro-survival signaling pathways mediating cardioprotection at a low level. However, the precise mechanisms for the dual role of ROS have not yet been fully clarified. To address this question, using intermittent hypobaric hypoxia (IHH) as a cardioprotective model, combining with H2O2 pre- and post-conditioning, we studied the level and roles of ROS during early reperfusion following ischemia in I/R injury and protection and explored the regulatory mechanisms underlying. Our results reveal that the elevated ROS generated during early reperfusion are injurious but insufficient to reach the threshold to trigger protective signaling pathways. The moderate level of ROS, higher than that elevated by I/R, during early reperfusion is critical for triggering cardioprotection against I/R injury via alleviating intracellular Ca2+ overload and preserving mitochondrial function through the efficient activation of Akt, PKCε[[Unable to Display Character: ]]and JAK2/STAT3 pathways. We then identified the downstream target of ROS-JAK2/STAT3 signals that interacts with the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2) to improve the activation of SERCA2 and subsequently regulate intracellular Ca2+ homeostasis. When the heart is exposed to excessive ROS, the activation of protective mechanisms reaches a plateau and fails to counteract the severe nonspecific oxidative stress. These findings provide a new angle to interpret the controversial roles of ROS in myocardial I/R and demonstrate that the differential effects of ROS in myocardial I/R are derived from a quantity-dependent wrestling between its detrimental and signaling roles.

Beneficial Impact of Temporary Portocaval Shunt in Living-Donor Liver Transplantation With a Difficult Total Hepatectomy

BackgroundAlthough a temporary portocaval shunt (TPCS) improves hemodynamic stability during liver transplantation, the role of TPCS is controversial. We assessed the effects of TPCS in patients undergoing living-donor liver transplantation (LDLT) with a difficult total hepatectomy. MethodsWe analyzed outcomes by means of retrospective review of 116 LDLTs performed in our institution from May 2011 to October 2013; among these, 33 recipients received TPCS (group I) and 83 did not (group II). We performed TPCS in a high-risk group, such as those with severe perihepatic adhesions, severe retrohepatic adhesions to the vena cava, or massive bleeding during total hepatectomy. Patient demographics and intraoperative and postoperative variables were reviewed. ResultsNo significant differences were observed in the perioperative variables except intraoperative blood loss. The transfusion requirement and operative time in group I were similar to those in group II despite the higher blood loss and more complicated cases. Hemodynamic status and the vasopressor requirement during the operation were similar between the 2 groups. We also compared 2 subgroups to evaluate the effects of TPCS more precisely in the high-risk patients: subgroup A (Model for End-Stage Liver Disease score [MELD], >20) and subgroup B (MELD, ≤20). The intraoperative requirements for platelet concentrate and epinephrine during the early reperfusion phase in subgroup A were significantly lower than those in subgroup A without TPCS. ConclusionsTPCS was a safe and useful procedure to improve hemodynamic status and postoperative LDLT outcomes in high-risk and select patients.

Microvascular Responses to Ischemic Stroke following Splenectomy

Splenectomy is known to protect the brain against the tissue injury that results from ischemia and reperfusion (I/R). The mechanisms that underlie this neuroprotective effect of splenectomy remain poorly understood. An early response to brain I/R is the recruitment of inflammatory cells and platelets into the cerebral microvasculature. The objective of this study was to determine if splenectomy alters the recruitment of adherent leukocytes and platelets in cerebral microvessels in the early phase (24 hr) of reperfusion following ischemic stroke. Wild type C57Bl/6 mice underwent splenectomy or sham splenectomy two weeks prior to 45 min middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. After 24 h of reperfusion, intravital fluorescence video microscopy was used to monitor and quantify the adhesion of leukocytes (rhodamine‐G6 labeled) and platelets (CFSE‐labeled) in cerebral venules. Infarct volume and peripheral blood counts were also measured. Splenectomy attenuates the recruitment of adherent platelets and leukocytes in the cerebral microvasculature and reduces infarct volume. The increased blood platelet count observed following MCAO was reduced to control levels in splenectomized animals. Peripheral leukocyte count did not differ between the splenectomized and sham splenectomized mice. These results implicate the spleen in I/R‐induced brain inflammation, thrombogenesis, and tissue necrosis as early as 24 h after reperfusion (Supported by a Malcolm Feist Postdoctoral Fellowship)

Intra-arterial Administration of Papaverine during Mechanical Thrombectomy for Acute Ischemic Stroke

The use of stent retrievers for mechanical thrombectomy in acute ischemic stroke may induce significant vasospasm, which at the early phases of reperfusion may be crucial for rethrombosis of the recanalized vessel. We aimed to study whether the use of intra-arterial papaverine in selected cases of vasospasm was associated with improved cerebral perfusion, arterial reocclusion, or increased hemorrhagic complications. We retrospectively studied 9 consecutive patients with large artery acute occlusion, treated with stent retriever and intra-arterial papaverine. Onset to administration of intravenous recombinant tissue-plasminogen activator time, baseline National Institute of Health Stroke Scale, time to reperfusion, number of passes of the stent retriever, modified Rankin Scale score at discharge, postprocedural hemorrhage, onset to reperfusion time, papaverine dose, and thrombolysis in cerebral infarction grade were recorded in all patients. After papaverine administration, the caliber of the infused arteries and their flow was increased in all cases. In none of the treated cases a reocclusion occurred after papaverine infusion. In one of the studied patients (11%), a parenchymal bleeding occurred 36 hours postoperatively. This small study suggests that intra-arterial infusion of papaverine for the treatment of cerebral vasospasm after mechanical thrombectomy in acute ischemic stroke is effective and safe.

Upregulation of the GEF-H1 pathway after transient cerebral ischemia

The microtubule-dependent GEF-H1 pathway controls synaptic re-networking and overall gene expression via regulating cytoskeleton dynamics. Understanding this pathway after ischemia is essential to developing new therapies for neuronal function recovery. However, how the GEF-H1 pathway is regulated following transient cerebral ischemia remains unknown. This study employed a rat model of transient forebrain ischemia to investigate alterations of the GEF-H1 pathway using Western blotting, confocal and electron microscopy, dephosphorylation analysis, and pull-down assay. The GEF-H1 activity was significantly upregulated by: (i) dephosphorylation and (ii) translocation to synaptic membrane and nuclear structures during the early phase of reperfusion. GEF-H1 protein was then downregulated in the brain regions where neurons were destined to undergo delayed neuronal death, but markedly upregulated in neurons that were resistant to the same episode of cerebral ischemia. Consistently, GTP-RhoA, a GEF-H1 substrate, was significantly upregulated after brain ischemia. Electron microscopy further showed that neuronal microtubules were persistently depolymerized in the brain region where GEF-H1 protein was downregulated after brain ischemia. The results demonstrate that the GEF-H1 activity is significantly upregulated in both vulnerable and resistant brain regions in the early phase of reperfusion. However, GEF-H1 protein is downregulated in the vulnerable neurons but upregulated in the ischemic resistant neurons during the recovery phase after ischemia. The initial upregulation of GEF-H1 activity may contribute to excitotoxicity, whereas the late upregulation of GEF-H1 protein may promote neuroplasticity after brain ischemia.

Nlrp3 plays no role in acute cardiac infarction due to low cardiac expression

The Nlrp3 inflammasome is a protein complex consisting of NLRP3, ASC and caspase-1, that upon activation produces a potent pro-inflammatory cytokine, IL-1β. Evidence has accumulated that the Nlrp3 inflammasome contributes to delayed (>3 d) cardiac remodeling processes occurring following cardiac ischemia–reperfusion (I/R). However, from in vitro experiments it is controversial whether Nlrp3 also contributes to the more acute phase of infarct development [ 1 Zuurbier C.J. Jong W.M.C. Eerbeek O. Koeman A. Pulskens W.P. Butter L.M. et al. Deletion of the innate immune NLRP3 receptor abolishes cardiac ischemic preconditioning and is associated with decreased Il-6/STAT3 signaling. PLoS One. 2012; 7: e40643 Crossref PubMed Scopus (67) Google Scholar , 2 Sandanger O. Ranheim T. Vinge L.E. Bliksoen M. Alfsnes K. Finsen A.V. et al. The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia–reperfusion injury. Cardiovasc Res. 2013; 99: 164-174 Crossref PubMed Scopus (319) Google Scholar , 3 Jong W.M.C. Zuurbier C.J. A role for NLRP3 inflammasome in acute myocardial ischemia–reperfusion injury?. Cardiovasc Res. 2013; 99: 226 Crossref PubMed Scopus (5) Google Scholar , 4 Takahashi M. Nlrp3 inflammasome as a novel player in myocardial infarction. Int Heart J. 2014; 55: 101-105 Crossref PubMed Scopus (159) Google Scholar ]. Information on the in vivo effects of Nlrp3 deletion is currently lacking [ [4] Takahashi M. Nlrp3 inflammasome as a novel player in myocardial infarction. Int Heart J. 2014; 55: 101-105 Crossref PubMed Scopus (159) Google Scholar ]. This information is necessary in the decision whether Nlrp3 therapeutic agents should be developed for the early reperfusion phase of cardiac I/R.

Aged kidneys are refractory to ischemic postconditioning in a rat model

Background: Ischemic postconditioning (IPoC) is defined as a series of intermittent interruptions of blood flow in the early phase of reperfusion that mechanically alters the hydrodynamics of reperfusion and it attenuates renal damage after ischemia/reperfusion (I/R) injury in vivo. But all of these data had been obtained in adult populations and whether this protection was maintained in aging kidneys was unknown. The objective of this study was to establish whether the efficacy of IPoC is maintained in aging kidneys. Materials and methods: The aged (24-month-old) and young (3-month-old) Wistar rats were used. Rats were subjected to 45 min of renal ischemia, both with and without treatment with IPoC. Serum urea nitrogen and creatinine levels, histological examination and apoptosis were assessed at 24 h. Oxidative stress was evaluated and apoptosis-related molecules were studied by Western blotting. Results: In young rat kidneys, IPoC significantly attenuated the renal dysfunction and cell apoptosis induced by I/R. In contrast, IPoC failed to limit renal dysfunction, possibly a consequence of increased apoptosis in aged rat kidneys. Conclusions: Our data indicated that IPoC was ineffective in aged rat kidneys. These findings may have major implications in that severe apoptosis in aged kidneys might be refractory to anti-apoptotic effect by IPoC.

Mitochondrial DAMPs: Novel Markers for Hepatic Ischemia Reperfusion Injury.

Introduction. Liver transplantation is the only life-saving treatment for patients with end-stage liver disease. An unavoidable consequence of the transplantation is ischemia reperfusion injury (IRI), an immune driven inflammatory response provoked by cellular oxygen deprivation. The inflammatory response, which results from acute oxidative stress and consequent hepatocellular death during the early reperfusion phase, causes the release of damage-associated molecular patterns (DAMPs). Recent studies show that the release of mitochondrial DAMPs (MTDs) is directly linked with functionally important immune consequences and trauma. We investigated the potential of MTDs to be used as diagnose markers for IRI in vitro and in vivo. Methods. Rat and human hepatocytes (McA RH-7777, HepG2) were used in the in vitro models, which were undergoing 3 treatments, respectively: 1) Control group: cells were grown in normothermic (37°C) and normoxic (5% CO2) conditions in DMEM medium for 7 hours; 2) Ischemia group: cells were incubated in generic UW preservation solution (SPS-1) in ischemic (4°C, anoxic) conditions for 6 hours; 3) IR group: cells were incubated in ischemic conditions for 6 hours in SPS-1 and switched to DMEM in a 37°C and 5% CO2 environment for 1 hour. Lewis Rats were used in in vivo model of complete hepatic ischemia for 60 min and reperfusion for another 60 min. Rats were randomly allocated to two groups (n=3): 1) A control group undergoing sham operation; 2) Rats undergoing in vivo hepatic IRI. The quantities of mtDNA were measure by qPCR using primers for cytochrome B, cytochrome C oxidase subunit III, and NADH genes. Results and Conclusion. When compared with control group in vitro, both the ischemia and IR groups had significantly increases in the quantity of 3 mtDNA genes and the increase in IR group was higher than ischemia group. In the in vivo model, the IRI group displayed a significant increase of mtDNA over time. There was also an increase of mtDNA at 30min in control group, but this decreased to baseline after 60min and remained stable during the next 60min. Our results indicate that IRI can elicit a significant increase of MTDs both in vitro and in vivo suggesting that mtDNA, especially cytochrome B gene, may serve as a novel marker in hepatic IRI during liver transplantation.

Temporal changes in hepatic antioxidant enzyme activities after ischemia and reperfusion in a rat liver ischemia model: effect of dietary fish oil.

This study investigated the hypothesis that administration of tilapia fish oil diet would attenuate warm liver ischemia/reperfusion injury (IRI) and whether fish oil modulates prooxidant/antioxidant status. Male Wistar rats were subjected to 30 min of approximately 70% hepatic ischemia followed by 1, 12, and 24 h reperfusion. Rats were randomly divided into three groups: sham-operated group (SO), control-warm hepatic ischemia (WI) group, and Oil-WI group given tilapia oil for 3 weeks followed by liver IRI. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured in the plasma. Levels of thiobarbituric acid reactive substances (TBARS) and antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were measured in liver fractions. In the sham group, there was no enzymatic or histological change. I/R caused significant increase in serum AST, ALT, and tissue TBARS levels. As compared to the control group, animals treated with tilapia oil experienced a significant decrease (p < 0.05) in AST and ALT levels in reperfusion periods. Tissue TBARS levels in Oil-WI group were significantly (p < 0.05) reduced as compared to control group at 60 min after reperfusion. After ischemia, 1, 12, and 24 h of reperfusion, CAT, SOD, and GPx values were the lowest in the Oil-WI group and highest in the control group and were statistically significant (p < 0.05). Histological analysis also revealed that fish oil provided some protection compared with the control group. Tilapia oil exerts a protective effect during the early phase of reperfusion, and it modulates prooxidant/antioxidant status of rat liver subjected to warm IRI.

Roles of MAPKAPK-2 and HSP27 in the reduction of renal ischemia-reperfusion injury by ischemic postconditioning in rats.

Ischemic postconditioning is a procedure during which intermittent reperfusions are performed in the early phase of reperfusion to protect organs from ischemia/reperfusion injury. And in this study, we mainly investigated the injury-alleviative role of mitogen-activated protein kinase-activating protein kinase-2 (MAPKAPK-2) and heat shock protein 27 (HSP27) in renal ischemic reperfusion injury during the procedure of ischemic postconditioning. Sprague-Dawley rats were randomly divided into four groups. The injury models were prepared by clipping the left renal pedicle of rats after ligating the right renal pedicle for 60 min. In the ischemic postconditioning group, sequential reperfusions were done for 10 s and another ischemia for 10 s for six cycles after kidney ischemia for 60 min. In addition, the specific inhibitor SB203580 was injected through caudal vein before ischemia. Serum creatinine, blood urea nitrogen and the expression of HSP27 and MAPKAPK-2 were detected 1, 3, 6 and 24 h later after reperfusion. Furthermore, phosphorylation of HSP27 and MAPKAPK-2 protein contents, histological changes and apoptosis were compared 24 h later after reperfusion. Our data showed that ischemic postconditioning attenuated the renal dysfunction and cell apoptosis induced by I/R and increased phosphorylation of MAPKAPK-2 and HSP27. The results indicated that ischemic postconditioning decreased apoptosis and improved renal function. Taken together, it is suggested that the renal protective effect may be related to the levels of HSP27 and MAPKAPK-2 activation.

Su1912 μ Opioid Receptor Activation Protects Against Intestinal Ischemia-Reperfusion Injury in Mice Through Different Pathways During Early and Late Phase of Reperfusion

Background: Neuropeptide S (NPS) is expressed by gastrointestinal (GI) enteroendocrine cells and macrophages in rat and man. Polymorphisms of the NPS receptor are linked to increased risk of inflammatory bowel disease as well as motor and sensory disturbances of the gut, suggesting a role for NPS in GI disorders. Further knowledge of NPS effects on motility and inflammation is needed. Methods: Studies of motility were carried out in rats with electrodes implanted in the small bowel. NPS was infused IV for 60 min and effects on myoelectrical activity were recorded. Motility effects of NPS were further studied as luminal pressure changes in anaesthetized rats where the proximal small intestine with intact blood supply was perfused with saline for motility recordings. Tissue samples were obtained from rats for evaluation of gene expression and protein elaboration of inflammatory biomarkers. Muscle strips of the human stomach, small intestine and colon were used for pharmacological analysis in organ baths of motility responses to NPS. Localization of the NPS receptor was done with fluorescent-protein tagging using Cy3-NPS. Plasma levels of NPS were measured using ELISA in 5 healthy and 14 IBD patients before and after meal. Results: In conscious rats, NPS 1 nmol kg-1min-1 increased irregular spiking, 4 nmol kg-1min-1 reduced spiking and increased the MMC cycle length (P=0.005). In anesthetized rats, NPS 0.01-1 nmol kg-1min-1 dose-dependently reduced small bowel motility (P<0.001). In rats, NPS also increased the mRNA expression of iNOS, and CXCL1 and IL-1β at the protein level. In human, fluorescent protein-tagging showed the NPS receptor primarily to be present in enolase-positive nerve fibers, but some scattered also in muscle tissue. In organ baths, NPS 1-100 nM caused TTX-dependent relaxations of the smooth muscle. We found no detectable levels of NPS in plasma of neither healthy subjects nor IBD patients. Conclusion: NPS has a smooth muscle-relaxing effect in the GI tract and dampens myoelectrical and contractile motor activity of the small bowel. This is achieved primarily through an inhibition of nerve-mediated relaxation of smooth muscle cells. Circulating NPS levels in humans are undetectable in healthy subjects and in active IBD. This implies that NPS exerts its inhibitory function by a paracrine or neurocrine mechanism in the gut. NPS seems also capable of intervening with inflammation by increasing gut cytokines and iNOS expression.

Measurement of free radicals using electron paramagnetic resonance spectroscopy during open aorto-iliac arterial reconstruction.

BackgroundAortic cross-clamping during abdominal aortic aneurysm (AAA) open repair leads to development of ischemia-reperfusion injury. Electron paramagnetic resonance spectroscopy (EPR) spin-trapping is a valuable method of direct measurement of free radicals.The objective of the study was to evaluate the results of EPR as a direct method of free radical measurement and degree of inflammatory response in open operative treatment of patients with AAA and aorto-iliac occlusive disease (AIOD).Material/MethodsThe study was performed on a group of 32 patients with AAA and 25 patients with AIOD scheduled for open repair. Peripheral venous blood for EPR spectroscopy and for SOD, GPx, ox-LDL, Il-6, TNF-alfa, CRP, and HO-1 were harvested. Selected parameters were established accordingly to specified EPR and immunohistochemical methods and analyzed between groups by Mann-Whitney U test and Wilcoxon matched-pairs signed-ranks test with Bonferroni correction.ResultsFree radicals level was correlated with the time of the aortic cross-clamping after the reperfusion of he first and second leg in AAA (r=0.7; r=0.47). ox-LDL in AAA decreased 5 min after reperfusion of the first leg (32.99 U/L, range: 14.09–77.12) and 5 min after reperfusion of the second leg (26.75 U/L, range: 11.56–82.12) and 24 h after the operation (25.85 U/L, range: 14.29–49.70). HO-1concentration increased to above the level before intervention 24 h after surgery. The activities of GPx and SOD decreased 5 min after the first-leg reperfusion in AAA. Twenty-four hours after surgery, inflammatory markers increased in AAA to CRP was 14.76 ml/l (0.23–38.55), IL-6 was 141.22 pg/ml (84.3–591.03), TNF-alfa was 6.82 pg/ml (1.76–80.01) and AIOD: CRP was 18.44 mg/l (2.56–33.14), IL-6: 184.1 pg/ml (128.46–448.03), TNF-alfa was 7.74 pg/ml (1.74–74.74).ConclusionsEPR spin-trapping demonstrates temporarily elevated level of free radicals in early phase of reperfusion, leading to decrease antioxidants in AAA. Elevated free radical levels decreased 24 h after surgery due to various endogenous antioxidants and therapies.

Postconditioning of the small intestine: which is the most effective algorithm in a rat model?

BackgroundMesenteric ischemia is a serious clinical condition requiring immediate surgical intervention. The unavoidable ischemic-reperfusion (IR) injury may be ameliorated using the appropriate postconditioning protocol. The aim of the present study was to investigate the optimal postconditioning algorithm in a rat model of intestinal ischemic–reperfusion injury. Materials and methodsMale Wistar rats were randomized into five groups (n = 10), one sham-operated, one IR, and three postconditioned groups, each with different protocols. The animals were subjected to 60 min of mesenteric ischemia, followed by 60 min of reperfusion. Postconditioning was applied at the onset of reperfusion using three different algorithms. Arterial pressure and mucosal microcirculation were monitored throughout the experiment. Mesenteric pH was determined at the early phase of reperfusion. Blood and tissue samples were taken at the end of reperfusion for histologic evaluation, serum lactate dehydrogenase, serum creatine kinase, serum tumor necrosis factor-α, serum interleukin-6, detailed mucosal antioxidant, and scavenger capacity assays. ResultsThe shorter and intermediate length cycles of postconditioning enhanced mucosal microcirculation and redox state and significantly delayed the normalization of mesenteric pH. Furthermore, milder histopathologic lesions and lower concentrations of serum necroenzymes and proinflammatory cytokines were detected compared with the IR group. The protective effect of postconditioning using longer cycles could only be seen in a tendentious manner. ConclusionsIn a rat model of intestinal ischemia–reperfusion, the shorter and intermediate length cycles of postconditioning proved to be more effective than the use of longer cycles.

High dose infusion of activated protein C (rhAPC) fails to improve neuronal damage and cognitive deficit after global cerebral ischemia in rats

Purpose: Recent studies demonstrated anticoagulatory, antiinflammatory, antiapoptotic, and neuroprotective properties of activated protein C (APC) in rodent models of acute neurodegenerative diseases, suggesting APC as promising broad acting therapeutic agent. Unfortunately, continuous infusion of recombinant human APC (rhAPC) failed to improve brain damage following cardiac arrest in rats. The present study was designed to investigate the neuroprotective effect after global cerebral ischemia (GI) with an optimized infusion protocol. Methods: Rats were subjected to bilateral clip occlusion of the common carotid arteries (BCAO) and controlled hemorrhagic hypotension to 40mmHg for 14min and a subsequent 5h-infusion of rhAPC (2mg/kg bolus+6mg/kg/h continuous IV) or vehicle (0.9% NaCl). The dosage was calculated to maintain plasma hAPC activity at 150%. Cerebral inflammation, apoptosis and neuronal survival was determined at day 10. Results: rhAPC infusion did not influence cortical cerebral perfusion during reperfusion and failed to reduce neuronal cell loss, microglia activation, and caspase 3 activity. Conclusion: Even an optimized rhAPC infusion protocol designed to maintain a high level of APC plasma activity failed to improve the sequels following GI. Despite positive reports about protective effects of APC following, e.g., ischemic stroke, the present study supports the notion that infusion of APC during the early reperfusion phase does not result in sustained neuroprotection and fails to improve outcome after global cerebral ischemia.

Endothelin-1 Release during the Early Phase of Reperfusion Is a Mediator of Myocardial Reperfusion Injury

Purpose: In acute myocardial infarction, left ventricular (LV) unloading reduces endothelin-1 (ET-1) release. We tested that endogenous ET-1 released during acute myocardial infarction might mediate ischemia/reperfusion (I/R) injury by stimulating increased intracellular calcium concentration, [Ca²⁺]_i, and apoptosis. Methods: Rabbits were subjected to 1 h of coronary artery occlusion followed by 3 h of reperfusion. Unloading was initiated 15 min prior to reperfusion and was maintained during reperfusion. The control group was subjected to reperfusion. Animals were treated with ET-1 receptor antagonist BQ123. In parallel, isolated rabbit cardiomyocytes subjected to simulated I/R with or without ET-1 or BQ123, intracellular Ca²⁺ and cell death were assessed with flow cytometry. Results: LV unloading prior to reperfusion reduced myocardial ET-1 release at 2 h of reperfusion. Infarct size was reduced in unloaded and BQ123 groups versus controls. LV unloading and BQ123 treatment reduced the percentage of apoptotic cells associated with increases in Bcl-2 protein levels in ischemic regions. BQ123 reduced both ET-1-induced [Ca²⁺]_i increase and cell death for myocytes subjected to stimulated I/R. Conclusion: We propose that components of reperfusion injury involve ET-1 release which stimulates calcium overload and apoptosis. Intravenous ET-1 receptor blockade prior to reperfusion may be a protective adjunct to reperfusion therapy in acute myocardial infarction patients.

228 PACING POSTCONDITIONING PROTECTS THE HEART BY A PATHWAY INVOLVING NITRIC OXIDE AND NATRIURETIC PEPTIDES

<h3>Introduction</h3> Brief periods of ventricular pacing during the early reperfusion phase (pacing-induced postconditioning, (PPC)) have been shown to protect the heart against ischemia reperfusion injury. The aim of this study was to explore the role of natriuretic peptides (atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)), protein kinase G (PKG) and nitric oxide (NO) in the protective effect of PPC. <h3>Methods</h3> Isolated perfused (Langendorff) rat hearts (n=6) were subjected to 30 minutes coronary occlusion and 30 minutes reperfusion. PPC consisted of 3 episodes of 30 seconds ventricular pacing alternated with 30 seconds atrial pacing during early reperfusion. Studied were control with only ischemia and reperfusion, PPC, PPC in combination with selective blockers of the ANP (H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ)), BNP (isatin) and NO (L-nitro-arginine-methylester (L-name)). Another sets of animals were treated with the agonists of ANP (ANP), BNP (Rat BNP-32), NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) and PKG activator 8-(4-chlorophenylthio)-guanosine 39, 59-cyclic monophosphate (CPT) at the beginning of reperfusion. Hemodynamics were computed by a data acquisition program. Infarct size and area at risk were determined using TTC and blue dye. <h3>Results</h3> Ischemia and reperfusion resulted in a poor recovery of heart hemodynamics. PPC significantly (p&lt;0.03) improved cardiac hemodynamics and decreased the infarct size (p&lt;0.05). Isatin and L-name completely abrogated the protective effect of PPC. However, ODQ did affect the protection afforded by PPC. When applied exogenously Rat BNP-32, CPT and SNAP showed a significant (P&lt;0.03) recovery in cardiac hemodynamics and significantly (P&lt;0.05) decreased the infarct size. This protection is comparable to that produced by PPC. <h3>Conclusions</h3> Exogenous and endogenous BNP, NO and PKG protected the heart against ischemia reperfusion injury. However, ANP is not involved in this protection. Interaction between ROS and NO did not block PPC protection to the heart.

Perioperative release of pro-regenerative biochemical signals from human renal allografts subjected to ischemia–reperfusion injury

Complement-derived molecules modulate the intensity of renal ischemia-reperfusion injury and may lead to the generation of biochemical signals [such as stromal-derived factor-1 (SDF-1) or sphingosine-1-phosphate (S1P)], which stimulate tissue/organ regeneration after injury. We tested the association between perioperative C5b-9/membrane attack complex (MAC) levels and intensified erythrocyte lysis, and asked whether significant changes in the levels of pro-regenerative substances occur during the early phase of renal allograft reperfusion. Seventy-five recipients were enrolled and divided into the early, slow, and delayed graft function (DGF) groups. Perioperative blood samples were collected from the renal vein during consecutive minutes of reperfusion. Extracellular hemoglobin (eHb), albumin (plasma S1P transporter), 8-iPF2α-III isoprostane, SDF-1 and S1P concentrations were measured. Throughout the reperfusion period, erythrocyte lysis intensified and was most pronounced in the DGF group. However, perioperative eHb levels did not correlate significantly with C5b-9/MAC values, but rather with the intensity of oxidative stress. No significant changes were observed in S1P, its plasma transporter (albumin) or SDF-1 levels, which were relatively low in all groups throughout the reperfusion period. Our study therefore demonstrates that no known biochemical signal for bone marrow-derived stem cell mobilization is released from human renal allografts to the periphery during the early phase of reperfusion.

P14: Time dependent regulation of MIF by S-nitros(yl)ation reduces apoptosis in myocardial I/R-injury

Background Macrophage migration inhibitory factor (MIF) is cardioprotective in myocardial ischemia/reperfusion (I/R) injury. MIF reduces apoptosis by its extracellular chemokine-like functions and the inhibition of the JNK pathway via intracellular JAB 1 binding. Unbound intracellular MIF reduces oxidative stress via its oxidoreductase activity. S -Nitros(yl)ation of MIF after nitrite application in I/R injury is a phenomenon of the early phase of reperfusion. S -Nitros(yl)ation enhances MIF’s cardioprotective properties: increase of the enzyme-like activity, reduction of infarct sizes and apoptosis. S -Nitros(yl)ation of MIF and MIF/JAB 1 interaction both depend on MIF’s cysteine 81. Whether S -nitros(yl)ation of MIF influences MIF/JAB 1 interaction during different phases of reperfusion and whether this affects MIF’s intra-and extracellular properties, is unclear. Hypothesis S -Nitros(yl)ation of MIF regulates its binding to JAB 1, its localization and its consecutive signaling pathways differentially in the various phases of reperfusion. Methods/Results TUNEL staining of murine hearts after in vivo I/R injury showed reduced apoptosis after S -nitros(yl)ation of MIF (14.5 ± 1.0% vs. 8.0 ± 3.5%, p In early phase of reperfusion, MIF/JAB 1 interaction was reduced upon S -nitros(yl)ation (0.3 ± 0.06% vs. 0.11 ± 0.04%, n = 4, p n = 5). Intracellular MIF protects from ROS induced damage: aconitase acitivity was higher after S -nitros(yl)ation than in control (2.1 ± 0.5 mU/mg protein vs. 1.25 ± 0.5 mU/mg protein, n = 3, p After 4 h of reperfusion, S -nitros(yl)ation and its effect on MIF/JAB 1 interaction was vanished: MIF/JAB 1 interaction was not different in both groups (0.34 ± 0.09% vs. 0.32 ± 0,05%, n = 5, p = ns). MIF concentrations in coronary effluent were higher after S -nitros(yl)ation than in control (4.9 ± 2 ng/min/g heart vs. 0.7 ± 0.3 ng/min/g heart, n = 5) showing time-delayed MIF release. Conclusion S -Nitros(yl)ation of MIF in I/R-injury reduces the MIF/JAB 1 interaction and MIF’s secretion. This detains MIF in the cell during early phase of reperfusion, where MIF reduces oxidative stress. In late phase-when S -nitros(yl)ation of MIF has vanished-MIF is released to the extracellular space, where other signaling pathways can be activated. Disclosure Nothing to disclose.

Blocking Properdin, the Alternative Pathway, and Anaphylatoxin Receptors Ameliorates Renal Ischemia-Reperfusion Injury in Decay-Accelerating Factor and CD59 Double-Knockout Mice

Complement is implicated in the pathogenesis of ischemia-reperfusion injury (IRI). The activation pathway(s) and effector(s) of complement in IRI may be organ specific and remain to be fully characterized. We previously developed a renal IRI model in decay-accelerating factor (DAF) and CD59 double-knockout (DAF(-/-)CD59(-/-)) mice. In this study, we used this model to dissect the pathway(s) by which complement is activated in renal IRI and to evaluate whether C3aR- or C5aR-mediated inflammation or the membrane attack complex was pathogenic. We crossed DAF(-/-)CD59(-/-) mice with mice deficient in various complement components or receptors including C3, C4, factor B (fB), factor properdin (fP), mannose-binding lectin, C3aR, C5aR, or Ig and assessed renal IRI in the resulting mutant strains. We found that deletion of C3, fB, fP, C3aR, or C5aR significantly ameliorated renal IRI in DAF(-/-)CD59(-/-) mice, whereas deficiency of C4, Ig, or mannose-binding lectin had no effect. Treatment of DAF(-/-)CD59(-/-) mice with an anti-C5 mAb reduced renal IRI to a greater degree than did C5aR deficiency. We also generated and tested a function-blocking anti-mouse fP mAb and showed it to ameliorate renal IRI when given to DAF(-/-)CD59(-/-) mice 24 h before, but not 4 or 8 h after, ischemia/reperfusion. These results suggest that complement is activated via the alternative pathway during the early phase of reperfusion, and both anaphylatoxin-mediated inflammation and the membrane attack complex contribute to tissue injury. Further, they demonstrate a critical role for properdin and support its therapeutic targeting in renal IRI.

Effects of Everolimus on Oxidative Stress in Kidney Model of Ischemia/Reperfusion Injury

Background/Aims: Reactive oxygen species play an important role in the pathogenesis of kidney ischemia/reperfusion injury (IRI) which may be influenced by immunosuppressive therapy. Pertinent to this, we investigated the effects of the mTOR inhibitor everolimus on redox settings and the activity of the anti-oxidative system in kidneys exposed to IRI. Methods: C57BL/6 mice were subjected to IRI by clamping both renal pedicles for 45 min. Everolimus was applied in daily, subcutaneous doses (0.25 mg/kg body weight), starting 1 day before IRI induction. Both everolimus-treated and non-treated mice were sacrificed at several time points, starting 30 min and finishing 7 days after IRI induction. Markers of oxidation such as glutathione and NADPH levels and anti-oxidative enzyme activities were determined in the kidneys. Results: In comparison to both sham and non-treated animals, the treatment with everolimus resulted in an increased level of markers of oxidation, including a lower level of glutathione, increased level of oxidized glutathione and reduced level of NADPH. The activity of superoxide dismutase was reduced in both experimental groups, but the effects were less pronounced in everolimus-treated animals. In the early phase of reperfusion, everolimus-treated animals showed higher activity of glutathione reductase in comparison to non-treated animals, whereas the activities of glutathione peroxidase and catalase were generally similar. The treatment with everolimus significantly reduced heme oxygenase-1 expression and increased iNOS mRNA expression when compared to non-treated animals. Conclusion: Our data imply that everolimus treatment may decrease cytoprotective capacity in kidneys exposed to IRI due to promoted oxidative/nitrosative stress.