First Hour Of Reperfusion Research Articles

Comparison of Different Ex-Vivo Preservation Strategies on Cardiac Metabolism in an Animal Model of Donation after Circulatory Death.

Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C-18 °C-25 °C-35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.

Ischemic post-conditioning regulates early inflammation response via STAT3 pathway at reperfusion in a mouse model of myocardial infarction

The protection against reperfusion injury conferred by ischemic post-conditioning (IPoC) has been reported to rely on several signalization pathways like RISK and SAFE. However, their respective roles and the downstream-mechanisms involved are not fully understood S727 STAT3 has been proposed to be required for STAT3 translocation to mitochondria. However, in recent years, several groups, amongst which our group, have contested the reality of STAT3 translocation in mitochondria of cardiomyocytes. Revisiting STAT3-dependent mechanisms involved in IPoC thus looks required and could open up a new understand of how it can efficiently decrease infarct size in animal models. Decipher STAT3-dependent mechanisms by which IPOC protects myocardium against reperfusion injury. C57b mice were subjected to ischemia-reperfusion with or without IPoC in presence/absence of STAT3 inhibitor ( n = 8 per each group). Heart, PBMC and blood samples were analyzed to determined: infarct size, phosphorylation level of STAT3 (Y705 and S727), genes and proteins expression, cytokines and troponin levels, immune cell type populations. I/R induced STAT3 phosphorylation at residues Y705 and S727, while IPoC mostly enhanced phosphorylation Y705-STAT3 residue within 15 minutes of reperfusion in the area-at-risk. No significant change in S727-STAT3 phosphorylation was detected. Inhibition of Y705-STAT3 phosphorylation increased by Stattic treatment prevented the cardioprotective effect of IPoC on the infarct size. Gene analysis in the area-at-risk revealed clusters of genes involved in wound healing and inflammation at 3 h after reperfusion. Screening of immune cells in myocardium 24 h after reperfusion revealed a decrease in neutrophils and macrophages, in the line of the decrease in infarct size. However, we noteworthily identified that IPoC was associated with an increase in a subpopulation of macrophages or dendritic cells that is not detected after Stattic treatment 24 h after reperfusion. Phosphorylation of Y705 STAT3 is critical for the protection induced by IPoC against reperfusion injury and it could, non-exhaustively, rely on the modulation of the inflammatory response during the first hours of reperfusion.

PPARβ/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia–reperfusion injury

BackgroundMesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARβ/δ (Peroxisome proliferator-activated receptors β/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARβ/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARβ/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated.ObjectivesThe aim of this study was to investigate the role of PPARβ/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction.Methods and resultsNaïve MSC and MSC pharmacologically activated or inhibited for PPARβ/δ were challenged with H2O2. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARβ/δ agonist GW0742 versus naïve MSC. In addition, PPARβ/δ-priming allowed to reveal the anti-apoptotic effect of MSC on cardiomyocytes and endothelial cells in vitro. When injected during reperfusion, in an ex vivo heart model of myocardial infarction, 3.75 × 105 PPARβ/δ-primed MSC/heart provided the same cardioprotective efficiency than 7.5 × 105 naïve MSC, identified as the optimal dose in our experimental model. This enhanced short-term cardioprotective effect was associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 h of reperfusion. By contrast, PPARβ/δ inhibition in MSC before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects.ConclusionAltogether these results revealed that PPARβ/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARβ/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the therapeutic injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Limiting calcium overload after cardiac arrest: The role of human albumin in controlled automated reperfusion of the whole body.

Regarding the overall inadequate results after cardiopulmonary resuscitation, the development of new treatment concepts is urgently needed. Controlled Automated Reperfusion of the whoLe body (CARL) represents a therapy bundle to control the conditions of reperfusion and the composition of the reperfusate after cardiac arrest (CA). The aim of this study was to investigate the plasma expander's role in the CARL priming solution and examine its mechanism of action. Viscosity, osmolality, colloid osmotic pressure (COP), pH and calcium binding of different priming solutions were measured in vitro and compared to in vivo data. N = 16 pigs were allocated to receive CARL following 20 min of untreated CA with either human albumin 20% (HA, N = 8) or gelatin polysuccinate 4% (GP, N = 8). Blood gas analyses were performed during the first hour of reperfusion and catecholamine and fluid requirements were recorded. Neurological outcome was assessed by neurological deficit scoring (NDS) on the seventh day. In vitro, addition of HA to the CARL priming solution resulted in higher COP and higher calcium-binding than GP. In vivo, treatment with HA led to greater reduction of ionized calcium and higher extracorporeal flows within the first 30 min of reperfusion with no difference in catecholamine support and fluid requirement. Seven-day survival of 75% with no difference in NDS was observed in both groups. Our data show that the plasma expander in the CARL priming solution has a significant effect on the initial reperfusate and can potentially influence the course of resuscitation. However, seven-day survival and NDS did not differ between groups.

Composition of Ex Vivo Lung Perfusion Solutions and Kinetics Define Differential Cytokine and Chemokine Secretion in a Porcine Cardiac Arrest Model of Lung Preservation

Purpose Ex vivo lung perfusion is an innovative technique to evaluate marginal lung organs especially after DCD. Normothermic continuous perfusion should reduce ischemic damage and improve the outcome of lung transplantation. However, the optimal protocol for EVLP has not been defined so far. The aim of our study was to compare cytokine/chemokine concentrations in perfusion solutions using different kinetics and solution compounds of EVLP in a porcine cardiac arrest model and to correlate the inflammatory parameters to oxygenation capacity values (∆pO2). Methods Following cardiac arrest and 1h of warm ischemia, lungs were harvested and flushed. Groups were processed as immediate (I-EVLP) 1h cold static preservation (CSP) and delayed (D-EVLP; 9h CSP). D-EVLP lungs were perfused with different solutions: Steen vs. modified Custodiol-N containing dextran (CD) or dextran/albumin (CDA). Cytokine/chemokine levels were analyzed at baseline (0h), after 1h, 4h using multiplex protein arrays. Results Concentrations of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α and IL-1β increased significantly (p Conclusion In a non-heart beating porcine cardiac arrest model with relevant IRI, lungs were perfused with normothermic EVLP. Longer CSP prior to EVLP did not result in enhanced cytokine secretion, but the first hours of reperfusion seem crucial for tissue damage. CD-solution dampens the cytokine/chemokine secretion probably by iron chelators and, possibly, protecting effects of dextran. Addition of albumin had no further effect on inflammation. Cytokine/chemokine concentrations correlated negatively with the oxygenation capacity, an important parameter for organ acceptance. These finding may help to optimize the ex vivo preservation procedure and possibly, more organs could reach the clinically relevant threshold for transplantation, thus, the pool of marginal donor lungs could be enlarged.

Dual Roles of Reactive Oxygen Species in Exacerbated Proinflammatory Effects of Caveolin‐2 Deficiency in Ischemia/Reperfusion and the Anti‐inflammatory Effects of Ethanol Preconditioning

Caveolin‐2 (Cav‐2), a member of caveolin protein family, is ubiquitously co‐expressed ubiquitously with caveolin‐1 (Cav‐1) in vascular endothelial cells. Cav‐2 interacts with Cav‐1 to form a hetero‐oligomeric complex within caveolae. In contrast with the extensively studied Cav‐1 including the functional role of Cav‐l expressed in ECs in ischemic injury, much less is known regarding the effects of Cav‐2. We hypothesized that (i) genetic deletion of Cav‐2 (CAV‐2 KO) would promote proinflammatory effects in the absence of I/R in wild‐type (WT) mice; (ii) postischemic inflammation will be exacerbated in CAV‐2 KO mice compared to WT animals; (iii) the proinflammatory effects induced by I/R are due to generation of reactive oxygen species (ROS) in CAV‐2 KO mice; and (iv) ingestion of ethanol at low to moderate levels (EPC) 24 hours prior to ischemia (45 min) followed by reperfusion (60 min) (I/R) will limit postischemic inflammation in CAV‐2 KO mice by a ROS‐dependent mechanism. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intes­tine. Prior to I/R, male CAV‐2 KO mice were treated with EPC, MnTBAP (cell‐permeable SOD mimetic) 1 hour prior to I/R, or MnTBAP 20 minutes prior to EPC, respectively, with postcapillary venular LR and LA quantified during the first hour of reperfusion. Our data shown that LR and LA were markedly elevated in male CAV‐2 KO mice under baseline condition relative to that noted in WT animals. I/R‐induced LR and LA were increased to a greater extent in CAV‐2 KO mice compared to WT animals. These postischemic increases in LR and LA were abolished by treatment with EPC or by exposure to MnTBAP 1 hour prior to I/R. MnTBAP pretreatment 20 minutes prior to EPC also abolished the protective effects of EPC. These results indicate that genetic deletion of CAV‐2 produces proinflammatory effects under baseline conditions and exacerbates these effects after I/R by a ROS‐dependent mechanism. Antecedent EPC prevents the proinflammatory responses to I/R in CAV‐2 KO mice. This protective effect of EPC was initiated by ROS.Support or Funding InformationNIH: R01‐AA022108, R01‐HL081860; DOD: W81XWH‐15‐1‐0624.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Compartment syndrome-induced muscle injury is diminished by the neutralization of pro-inflammatory cytokines.

Objectives:Compartment syndrome (CS) is one of the most devastating consequences of musculoskeletal trauma. The pathophysiology of CS includes elevation of intracompartmental pressure (ICP), causing damage to the microcirculation, decreased oxygen delivery, tissue anoxia, and cell death. CS is a combined ischemic and inflammatory condition that induces the systemic inflammatory cascade. In complete ischemia, within the first hour of reperfusion, a peak in the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α) has been previously reported. The purpose of this study was to examine the suspected systemic inflammatory cytokine/chemokine release in response to CS, and to evaluate the microvascular dysfunction, tissue injury, and inflammatory response following the neutralization of pro-inflammatory cytokines TNF-α and/or interleukin-1 beta (IL-1β).Methods:Twenty-eight male Wistar rats were randomly assigned into 5 groups: Sham (no CS), CS (with isotype control), CS+TNF-α neutralizing antibody (NA), CS+IL-1β NA, CS+Combo (both TNF-α and IL-1β NA). CS was induced by elevation of ICP above 30 mm Hg through an infusion of isotonic saline into the anterior compartment of the hind limb for 2 hours; NA were administered just prior to fasciotomy. Microvascular perfusion, cellular tissue injury, and inflammatory response within the extensor digitorum longus muscle were assessed using intravital video microscopy for 45 minutes after fasciotomy. Systemic levels of 24 different cytokines/chemokines were also measured, using the xMAP Luminex technology.Results:Of the 24 cytokines/chemokines sampled, 6 were significantly elevated from their baseline levels, and included the pro-inflammatory cytokines TNF-α, IL-1β, growth-related oncogene/keratinocyte chemoattractant (GRO/KC), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), and the anti-inflammatory cytokine IL-10. CS resulted in a significant decrease in microvascular perfusion, from 75 ± 2% continuously perfused capillaries in the sham to 31 ± 4% in CS (P < .001), a significant increase in tissue injury (0.33 ± 0.4 versus 0.04 ± 0.01 in sham) and leukocyte activation (14 ± 2 adherent leukocytes/1000 μm2 versus 2 ± 1 adherent leukocytes/100 μm2 in sham, P < .001). CS-associated tissue injury was significantly decreased with TNF-α neutralization (P < .05), both when administered alone or in combination with IL-1β (P < .05). Additionally, TNF-α neutralization blocked CS-associated leukocyte activation (P < .05); IL-1β neutralization also diminished leukocyte adhesion (P < .05). Perfusion remained virtually unchanged in CS animals treated with NA (36 ± 4%, 32 ± 3% and 30 ± 2% in CS+TNF-α, CS+IL-1β and CS+Combo groups, respectively).Conclusion:The results of this study indicate that CS induces a systemic inflammation, as evidenced by upregulation of inflammatory cytokines/chemokines in circulation. Neutralization of TNF-α led to a significant reduction in tissue injury; however, it had no effect on the CS-induced microvascular dysfunction. This suggests a distinct role of TNF-α in the pathophysiology of muscle injury in CS.

Rapamycin and fasting sustain autophagy response activated by ischemia/reperfusion injury and promote retinal ganglion cell survival

Autophagy, the cellular process responsible for degradation and recycling of cytoplasmic components through the autophagosomal–lysosomal pathway, is fundamental for neuronal homeostasis and its deregulation has been identified as a hallmark of neurodegeneration. Retinal hypoxic–ischemic events occur in several sight-treating disorders, such as central retinal artery occlusion, diabetic retinopathy, and glaucoma, leading to degeneration and loss of retinal ganglion cells. Here we analyzed the autophagic response in the retinas of mice subjected to ischemia induced by transient elevation of intraocular pressure, reporting a biphasic and reperfusion time-dependent modulation of the process. Ischemic insult triggered in the retina an acute induction of autophagy that lasted during the first hours of reperfusion. This early upregulation of the autophagic flux limited RGC death, as demonstrated by the increased neuronal loss observed in mice with genetic impairment of basal autophagy owing to heterozygous ablation of the autophagy-positive modulator Ambra1 (Ambra1+/gt). Upregulation of autophagy was exhausted 24 h after the ischemic event and reduced autophagosomal turnover was associated with build up of the autophagic substrate SQSTM-1/p62, decreased ATG12-ATG5 conjugate, ATG4 and BECN1/Beclin1 expression. Animal fasting or subchronic systemic treatment with rapamycin sustained and prolonged autophagy activation and improved RGC survival, providing proof of principle for autophagy induction as a potential therapeutic strategy in retinal neurodegenerative conditions associated with hypoxic/ischemic stresses.

HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

RationaleRecent evidence indicates that histone deacetylase enzymes (HDACs) contribute to ischemia reperfusion (I/R) injury, and pan-HDAC inhibitors have been shown to be cardioprotective when administered either before an ischemic insult or during reperfusion. We have shown previously that selective inhibition of class I HDACs provides superior cardioprotection when compared to pan-HDAC inhibition in a pretreatment model, but selective class I HDAC inhibition has not been tested during reperfusion, and specific targets of class I HDACs in I/R injury have not been identified. ObjectiveWe hypothesized that selective inhibition of class I HDACs with the drug MS-275 (entinostat) during reperfusion would improve recovery from I/R injury in the first hour of reperfusion. Methods and resultsHearts from male Sprague-Dawley rats were subjected to ex vivo I/R injury±MS-275 class I HDAC inhibition during reperfusion alone. MS-275 significantly attenuated I/R injury, as indicated by improved LV function and tissue viability at the end of reperfusion. Unexpectedly, we observed that HDAC1 is present in the mitochondria of cardiac myocytes, but not fibroblasts or endothelial cells. We then designed mitochondria-restricted and mitochondria-excluded HDAC inhibitors, and tested both in our ex vivo I/R model. The selective inhibition of mitochondrial HDAC1 attenuated I/R injury to the same extent as MS-275, whereas the mitochondrial-excluded inhibitor did not. Further assays demonstrated that these effects are attributable to a decrease in SDHA activity and subsequent metabolic ROS production in reperfusion. ConclusionsWe demonstrate for the first time that HDAC1 is present within the mitochondria of cardiac myocytes, and mitochondrial HDAC1 contributes significantly to I/R injury within the first hour of reperfusion.

Different response to antiepileptic drugs according to the type of epileptic events in a neonatal ischemia-reperfusion model

Perinatal arterial stroke is the most frequent form of cerebral infarction in children. Neonatal seizures are the most frequent symptom during the neonatal period. The current management of perinatal stroke is based on supportive care. It is currently unknown if treatment of the seizures modifies the outcome, and no clinical studies have focused on seizures during neonatal stroke. We studied the effect of phenobarbital and levetiracetam on an ischemic-reperfusion stroke model in P7 rats using prolonged electroencephalographic recordings and a histologic analysis of the brain (24h after injury). The following two types of epileptic events were observed: 1) bursts of high amplitude spikes during ischemia and the first hours of reperfusion and 2) organized seizures consisting in discharges of a 1–2Hz spike-and-wave. Both phenobarbital and levetiracetam decreased the total duration of the bursts of high amplitude spikes. Phenobarbital also delayed the start of seizures without changing the total duration of epileptic discharges. The markedly limited efficacy of the antiepileptic drugs studied in our neonatal stroke rat model is frequently observed in human neonatal seizures. Both drugs did not modify the stroke volume, which suggests that the modification of the quantity of bursts of high amplitude spikes does not influence the infarct size. In the absence of a reduction in seizure burden by the antiepileptic drugs, we increased the seizure burden and stroke volume by combining our neonatal stroke model with a lithium-pilocarpine-induced status epilepticus. Our data suggest that the reduction of burst of spikes did not influence the stroke volume. The presence of organized seizure with a pattern close to what is observed in human newborns seems related to the presence of the infarct. Further research is required to determine the relationship between seizure burden and infarct volume.

Abstract 301: Initial Increase of Klf5 and Ppara Expression After Myocardial Ischemia/Reperfusion in Mice Appears to be Critical for Survival

Krüppel-like factors (KLF) have important roles in metabolism. We previously found that KLF5 is a positive transcriptional regulator of peroxisome proliferator-activated receptor α ( Ppara) , a central regulator of cardiac fatty acid oxidation (FAO). Mice with cardiomyocyte-specific Klf5 ablation ( α MHC-Klf5 -/- ) had reduced cardiac Ppara expression and FAO. At age 6-12 months these mice develop distinct cardiac dysfunction. The role of PPARα activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to study if Ppara expression changes during I/R are driven by KLF5 and explore its protective or detrimental role. Wild type mice were subjected to in vivo I/R or sham surgery. I/R resulted in an initial increase in Ppara , and its target gene pyruvate dehydrogenase kinase 4 ( Pdk4) mRNA after 2h reperfusion, followed by decreased expression after 24h reperfusion. The Ppara expression is associated with parallel changes in cardiac Klf5 mRNA expression. Concurrent, there was a decrease of cardiac FAO-related genes carnitine palmitoyl-transferase 1β ( Cpt1b), very long chain acyl-CoA dehydrogenase (Vlcad), and acyl-CoA oxidase ( Aox) in mice with I/R. To define the cell type causing the temporal changes in Klf5 and Ppara after I/R we isolated primary cardiomyocytes and fibroblasts. Our data suggest a similar effect in primary isolated cardiomyocytes only. Klf5 mRNA expression is increased after 2 hour hypoxia and normalized after 4 hour re-oxygenation in cardiomyocytes, whereas there are no changes after hypoxia/normoxia in fibroblasts. To assess the importance of cardiomyocyte KLF5 in I/R we used α MHC-Klf5 -/- mice. Interestingly, despite reduced FAO, 7 month old αMHC-Klf5 -/- mice subjected to I/R had a marked increase in mortality; 4 of 7 αMHC-Klf5 -/- mice died within the first 24h of reperfusion while no mortality was observed in age-matched wild type mice that underwent I/R. In conclusion, I/R is associated with an increase in Klf5 and Ppara in the first hours of reperfusion followed by a decrease in Klf5 and Ppara , likely accounted for by cardiomyocytes. Increased mortality for α MHC-Klf5 -/- mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival.

Intestinal ischemia–reperfusion induced diaphragm contractility dysfunction: Electrophysiological and ultrastructural study in a neonatal rat model

AimTo evaluate the remote effect of intestinal ischemia reperfusion (IR) injury mediated by tumor necrosis factor alpha (TNF-α) on diaphragm contractility functions and whether administration of NAC may counteract the possible detrimental effects in an experimental neonatal rat model. Methods40 Wistar rat pups were randomized into four groups; ten animals in each. Intestinal ischemia was conducted by obstructing mesentery of intestines by a silk loop. In the control group; only laparotomy was performed. After 1h ischemia, reperfusion was conducted for 1h in 1h group, 24h for 24h group and 24h for 24h+NAC group but administration of NAC (150mg/kg/day) intraperitoneally twice a day was performed. Inflammatory response was evaluated by tissue TNF-α level and contractility functions by mechanic activity studies of the diaphragm. Electrophysiology of the diaphragm and the phrenic nerve was conducted to determine neuropathy or myopathy and transmission electron microscopy was performed to evaluate ultrastructural changes in the phrenic nerve. ResultsDiaphragm tissue TNF-α level significantly increased in 1h and 24h groups (P=0.004, P=0.0001; respectively). Diaphragm mechanic activation force and duration significantly decreased at 1h and 24h (P=0.004, P=0.02 and P=0.0001, P=0.0001; respectively). NAC administration significantly prevented decrease in the maximal contraction and the duration (P<0.001). Phrenic nerve compound action potential (CMAP) amplitude significantly decreased in 1h group (P<0.0001) and NAC administration significantly prevented this decrease when compared with 24h group (P<0.001). In diaphragmatic needle electromyography, the duration of motor unit potentials (MUP) was prolonged significantly when compared with control group. Contractility and electrophysiological studies were indicating primarily neuropathy in diaphragm dysfunction. Histopathology revealed axonal and myelin degeneration in the 1h and 24h group, but less injury in the NAC administered group. ConclusionsIntestinal IR induced elevation of TNF-α level in the diaphragm. Impairment in the diaphragm contractility and neuropathic changes in the phrenic nerve occurred even in the first hour of reperfusion. NAC administration prevented these detrimental effects.

Abstract 113: Klf5 and Ppara Expression is Increased at the Early Stage and Reduced at the Late Stage of Myocardial Ischemia/Reperfusion in Mice

Inhibition of cardiac fatty acid oxidation (FAO) is considered beneficial after ischemia reperfusion (I/R). Krüppel-like factors (KLF) have an important role in metabolism. In a model of cardiac energetic deficiency (LPS-treated mice), in silico promoter analysis and whole genome array analysis indicated cardiac KLF5 as important regulator of Ppara . Gene expression analysis in human ventricular myocytes (AC16) treated with Ad-Klf5 followed by ChIP analysis confirmed that KLF5 is a positive transcriptional regulator of Ppara . Mice with cardiomyocyte-specific Klf5 ablation (αMHC-Klf5 -/- ) that we made had reduced cardiac Ppara expression and other FAO-related genes. The role of PPARα activation in I/R injury is unclear as both beneficial and detrimental effects have been reported. We aimed to gain insight in the effects of KLF5 and PPARα on acute I/R injury. To mimic I/R in vitro , AC16 cells were subjected to hypoxia (9.5 h) followed by short (1h) or prolonged (14h) normoxia. Ppara expression was initially (1h) increased (p&lt;0.05) and then (14 h) decreased (p&lt;0.05). Klf5 expression pattern showed a trend of similar changes . Infarction was performed by 30 min of left coronary artery occlusion followed by 2 or 24 hours of reperfusion in wild type mice. I/R resulted in a trend for an initial increase in Klf5 (2-fold, p=0.05), Ppara (3-fold, p=0.08), and Pdk4 (8-fold, p&lt;0.01) mRNA at 2h post-surgery. Klf5 (2-fold; p&lt;0.01) and Ppara (9-fold; p&lt;0.05) expression were decreased 24h post-surgery. Consistent with Ppara changes, there was a 2-fold decrease of Cpt1 (p&lt;0.01) and Vlcad (p&lt;0.01) and a trend for decreased Aox (2-fold), Lcad (2-fold), and Pdk4 (6-fold) compared to sham. Echocardiography indicated normal cardiac function after 24h post-surgery. Despite reduced FAO, αMHC-Klf5 -/- mice subjected to I/R had a marked increase in mortality; 40% (4 of 10) of αMHC-Klf5 -/- mice died within the first 24h of reperfusion while no mortality was observed in wild type mice that underwent I/R. In conclusion, I/R is associated with an increase in Klf5 and Ppara in the first hours of reperfusion followed by a decrease in Klf5 and Ppara . Increased mortality for αMHC-Klf5 -/- mice with I/R injury suggests that the initial increase may be an adaptive response that is critical for survival.

Intravenous Superoxide Dismutase Administration Reduces Contralateral Lung Injury Induced by Unilateral Lung Ischemia and Reperfusion in Rats Through Suppression of Activity and Protein Expression of Matrix Metalloproteases

BackgroundIschemia and reperfusion (I/R) of the lungs induces massive superoxide radical production. On the other hand, matrix metalloproteases (MMPs) were shown to play an essential role in I/R-associated lung injury. We aimed to investigate the lung-protective efficacy of intravenous superoxide dismutase (SOD) administration and its relation with MMPs activity in the lungs subsequent to I/R injury. MethodsTwenty-two male Sprague-Dawley rats were divided into a sham group (n = 6), a unilateral lung I/R group (n = 8), and a SOD-treated lung I/R group (n = 8). Unilateral lung ischemia was conducted by occluding the left lung hilum for 90 min, followed by 5 hours of reperfusion through release of the occlusion. In the SOD-treated group, SOD was administered intravenously during the first hour of reperfusion. We assessed the protein contents in the broncho-alveolar lavage fluid (PCBAL) as a marker for protein permeability and lung wet-to-dry weight ratio (W/D) for lung water content. We also measured levels of lipid peroxidation and MMP activity in the lungs, by tissue malonedealdehyde (MDA) level with the use of enzyme-linked immunoassay, and the gelatin zymography technique, respectively. ResultsForty-eight hours of left-lung I/R significantly increased PCBAL (P < .001), W/D (P < .05), tissue MDA level (P < .05), and MMP-9 and MMP-2 activity. SOD treatment attenuated I/R-induced contralateral lung injury, reducing pulmonary permeability, lipid peroxidation, and MMP activities. ConclusionsI/R injury of the left lung induced increases in W/D, PCBAL, MDA level, and MMP-9 activity in the right lung. SOD treatment during the first hour of a 5-hour reperfusion protected the lung through suppressing MMP-9 activity and reducing tissue lipid peroxidation.

Late anti‐apoptotic effect of KATP channel opening in skeletal muscle

Necrosis and apoptosis caused by ischaemia-reperfusion (IR) result in myocyte death and atrophy. ATP-sensitive K(+) (K(ATP) ) channels activation increases tissue tolerance of IR-injury. Thus, in the present study, we evaluated the effects of K(ATP) channel activation on skeletal muscle apoptosis after IR. Male Wistar rats were treated with 40mg/kg, i.p., diazoxide (a K(ATP) channel opener) or 5mg/kg, i.p., glibenclamide (a K(ATP) channel inhibitor) 30min before the induction of 3h ischaemia, followed by 6, 24 or 48h reperfusion. At the end of the reperfusion period, the gastrocnemius muscle was removed for the analysis of tissue malondialdehyde content, superoxide dismutase (SOD) and catalase (CAT) activity, Bax and Bcl-2 protein expression, histological damage and the number of apoptotic nuclei. Ischaemia-reperfusion increased malondialdehyde content (P<0.01) and Bax expression (P<0.01) and induced severe histological damage, in addition to decreasing CAT and SOD activity (P<0.01 and P<0.05, respectively) and Bcl-2 expression (P<0.01). Diazoxide reversed the effects of IR on tissue damage, MDA content, SOD and CAT activity (after 6 and 24h reperfusion; P<0.05) and Bax and Bcl-2 expression (after 24 and 48h reperfusion; P<0.01). In contrast, glibenclamide pretreatment had no effect. The number of apoptotic nuclei in the IR and glibenclamide-pretreated groups increased significantly (P<0.001 vs Sham). In contrast, diazoxide pretreatment decreased the number of apoptotic nuclei compared with the IR group (P<0.01). The results of the present study suggest that the K(ATP) channel activator diazoxide attenuates lipid peroxidation during the first hour of reperfusion and modulates apoptotic pathways at later time points.

Ischemic postconditioning regulates cardiomyocyte autophagic activity following ischemia/reperfusion injury.

Ischemic postconditioning (IPostC) is a promising protective mechanism for combating reperfusion injury. However, the role of autophagy in the protective effects of IPostC and the associated signaling pathways have remained to be elucidated. Male Sprague Dawley rats were subjected to 30 min ischemia and 1, 2, 3, 6, 12 and 24 h of reperfusion, with or without IPostC treatment. Autophagic flux was evaluated by detecting mRNA and protein expression levels of microtubule associated protein 1 light chain 3 and p62. Phosphorylated (p)-P70S6 kinase (P70S6K), p-adenosine monophosphate-activated kinase (AMPK) and Beclin 1 protein levels were measured by western blot analysis. Myocardial infarct size was measured using staining with Evans blue dye and myocardial apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Autophagic activity was observed to be inhibited within the first hour of reperfusion, increased during 2-6 h and reduced from 12-24 h following IPostC compared with reperfusion without IPostC. Inhibition of autophagy by chloroquine significantly reversed the effects of IPostC on myocardial infarct size and the levels of apoptosis. IPostC significantly increased Beclin 1 levels, inhibited AMPK activation and increased P70S6K activation within the first hour compared with reperfusion without IPostC. IPostC attenuated the upregulation of Beclin 1 levels, increased AMPK activation and reduced P70S6K activation between 2 and 12 h, and subsequently exerted the opposite effects on these molecules between 12 and 24 h. IPostC was demonstrated to regulate autophagic activity in a time‑dependent manner. The Beclin 1 and AMPK-mammalian target of rapamycin signaling pathways are suggested to be involved in the regulation of IPostC in autophagy.

Effects of α-MSH on ischemia/reperfusion injury in the rat sciatic nerve

Background:Ischemia/reperfusion (I/R) causes the production of toxic free radicals and leads to pathological changes in nerve tissue. We investigated the effect of alpha-melanocyte stimulating hormone (α-MSH) in a rat model for sciatic nerve I/R and discuss the possible cytoprotective and antioxidant mechanism of α-MSH against ischemic fiber degeneration.Methods:Experiments were performed using 42 adult male Wistar rats. Rats were divided into six experimental groups: control group, ischemia group, I/R groups, and α-MSH treated groups. Ischemia was produced by clamping of the femoral vessels. Immediately after ischemia that lasted 3 h, 75 μg/kg of α-MSH was administered subcutaneously before reperfusion and the tissue malondialdehyde (MDA) level was evaluated as an indicator of lipid peroxidation in groups with different reperfusion periods.Results:The reperfusion injury did not begin in the first hour of reperfusion after 3 h of ischemia, and MDA levels increased on the first day of reperfusion. During the first day, blood MDA levels were decreased in the α-MSH group compared to the control group. The tissue from animals pre-treated with α-MSH showed fewer morphological alterations. Myelin breakdown was significantly diminished after treatment with α-MSH, and the ultrastructural features of axons showed remarkable improvement. Two-way analysis of variance was used for comparing three or more groups. When a significant difference existed, the post-hoc multiple-comparison test was applied to demonstrate the differences.Conclusions:The results confirm that pre-treatment with α-MSH after ischemia protected the peripheral nerves against I/R injury.

Different neuroprotective responses of Ginkgolide B and bilobalide, the two Ginkgo components, in ischemic rats with hyperglycemia

Ginkgo biloba extracts show neuroprotective effects during cerebral ischemia, but with various components, the mechanisms of action remain unclear. In this study, we tested the effects of Ginkgolide B (GB) and bilobalide (BB) on normoglycemic and hyperglycemic rats subjected to transient cerebral ischemia. Rats were administered p.o. with different Ginkgo components GB (6mg/kg) or BB (6mg/kg) once daily for 7days. Hyperglycemia was made by jugular vein infusion of glucose and transient middle cerebral artery occlusion/reperfusion was induced by a suture insertion technique. Results showed that both GB and BB exerted neuroprotection under normoglycemia, as determined by infarct volume and neurological deficit scores. Yet, BB showed less protective effects during hyperglycemic cerebral ischemia. Cerebral blood flow (CBF) was evaluated during occlusion and the first hour of reperfusion. BB but not GB caused acute increase in CBF after reperfusion, especially in hyperglycemia. Reactive oxygen species and malondialdehyde levels were reduced by GB in both models but BB were not effective in reactive oxygen species or malondialdehyde control in hyperglycemia ischemic rats. These results suggested that CBF plays crucial roles during early stage of reperfusion in the presence of hyperglycemia. Administration of compound that improves CBF may have little effect in hyperglycemic stroke.

Abstract P287: Therapeutic Hypothermia Cardioprotection During Cardiac Arrest Alters Reperfusion-Associated mTORC1 and AMPK Signaling

Introduction: Therapeutic hypothermia (TH) is highly cardioprotective in cellular and animal models of ischemia/reperfusion (I/R) and cardiac arrest and is dependent upon Akt. The mammalian target of rapamycin complex 1 (mTORC1) is a downstream target of Akt and an important regulator of cellular energy utilization and survival but its relationship to TH is not clear. We hypothesized that TH attenuates mTORC1 activation while simultaneously activating the cell survival kinases AMP activated protein kinase (AMPK) and Stat3. We further hypothesized that inhibitors of mTORC1 or activators of AMPK would replicate and/or enhance TH cardioprotection. Methods: Cardiomyocytes isolated from 1-2-day old C57BL6/J mice, were exposed to simulated I (90 min)/R (3 h). For TH, cells were cooled from 37C to 32C during ischemia and the first hour of reperfusion. Heart samples were also obtained from C57BL/6 mice that underwent an 8-min cardiac arrest. Following 6 min, the mice were randomized to normothermia (NT, 37C) or TH (30C) extended during CPR and for 1 h after resuscitation. Protein lysates were collected at serial time points for Western blot analyses. Results: Cardiomyocyte death during ischemia was minimal, but accelerated during reperfusion (4% vs. 46%,p&lt;.05 n=5). TH attenuated this cell death (24% p&lt;.05 n=5) and attenuated reperfusion-induced mTOR activation seen within 30 min as measured by differential phosphorylation of the mTOR targets 70S6K and eEF2. Compared to NT, TH-treated cardiomyocytes and hearts demonstrated increased p-AMPK after resuscitation, consistent with mTOR inhibition. Stat3 also showed enhanced phosphorylation on tyrosine 705. The mTOR inhibitor rapamycin (100nM) given during I/R similar to TH affected 70S6K and eEF2 and significantly reduced cell death (28% vs. 42%, p&lt;.05 n=5). The AMPK activator metformin (100uM) also significantly reduced cell death (22%, p&lt;.05 n=4). Conclusion: TH cardioprotection is associated with altered signaling events suggesting mTORC1 inhibition and activation of the cell survival kinases AMPK and Stat3. Pharmacologic strategies that mimic these TH effects are available and could be useful as TH adjuncts or mimetics that improve survival after cardiac arrest.

Abstract 264: Therapeutic Hypothermia Cardioprotection Is Associated with Enhanced pAMPK but Not ATP Preservation

Introduction: Therapeutic Hypothermia (TH) is cardioprotective following ischemia/reperfusion (I/R) injury and improves survival following cardiac arrest in an Akt dependent manner. AMP activated protein kinase (AMPK) is a highly conserved, cardioprotective, sensor of cellular energetic stress and ATP depletion that is activated by ischemia. Phosphorylated AMPK (pAMPK) is also a significant inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) that is activated by Akt but inhibited by TH. In this study we hypothesized that TH enhances pAMPK independent of cellular ATP while increasing cell survival. We further hypothesized that strategies to enhance pAMPK would mimic TH cardioprotection by inhibiting mTORC1. Methods: Cardiomyocytes isolated from 1-2-day old C57BL6/J mice, were exposed to simulated Ischemia (90 min) followed by reperfusion. For TH, cells were cooled from 37C to 32C during ischemia and the first hour of reperfusion. Protein lysates were collected at serial time points for changes in pAMPK by western blot analyses. ATP measurements were made using a standard bioluminescence assay. Results: pAMPK was significantly increased during ischemia as compared to controls. During reperfusion, pAMPK decreased but was maintained by TH (n=5, p&lt;.05). Total AMPK was unchanged. Total cellular ATP decreased significantly with ischemia and TH to 6% and 13% of controls (n=5, p&lt;.05). Following 30 minutes of reperfusion, ATP increased to 9% (IR) and 26% (TH) of controls. Although total cellular ATP increased with TH, these changes were not significant and remained substantially reduced from controls. Cardiomyocyte cell death during ischemia was minimal but increased during reperfusion (4% vs. 46%, p.05, n=5). This cell death was attenuated by TH (24%, p&lt;.05, n=5). The pAMPK activator metformin (100um) similarly attenuated reperfusion associated cell death (22%, p&lt;.05, n=4) while inhibiting mTORC1. Conclusions: TH is associated with enhanced cardiomyocyte survival and pAMPK following IR. These events were not associated with enhanced ATP preservation during ischemia. Pharmacologic strategies to mimic TH effects on AMPK may be beneficial to enhance survival following cardiac arrest.