Hypothermic Ischemia-reperfusion Research Articles

CIRP attenuates acute kidney injury after hypothermic cardiovascular surgery by inhibiting PHD3/HIF-1α-mediated ROS-TGF-β1/p38 MAPK activation and mitochondrial apoptotic pathways

BackgroundThe ischemia–reperfusion (IR) environment during deep hypothermic circulatory arrest (DHCA) cardiovascular surgery is a major cause of acute kidney injury (AKI), which lacks preventive measure and treatment. It was reported that cold inducible RNA-binding protein (CIRP) can be induced under hypoxic and hypothermic stress and may have a protective effect on multiple organs. The purpose of this study was to investigate whether CIRP could exert renoprotective effect during hypothermic IR and the potential mechanisms.MethodsUtilizing RNA-sequencing, we compared the differences in gene expression between Cirp knockout rats and wild-type rats after DHCA and screened the possible mechanisms. Then, we established the hypothermic oxygen–glucose deprivation (OGD) model using HK-2 cells transfected with siRNA to verify the downstream pathways and explore potential pharmacological approach. The effects of CIRP and enarodustat (JTZ-951) on renal IR injury (IRI) were investigated in vivo and in vitro using multiple levels of pathological and molecular biological experiments.ResultsWe discovered that Cirp knockout significantly upregulated rat Phd3 expression, which is the key regulator of HIF-1α, thereby inhibiting HIF-1α after DHCA. In addition, deletion of Cirp in rat model promoted apoptosis and aggravated renal injury by reactive oxygen species (ROS) accumulation and significant activation of the TGF-β1/p38 MAPK inflammatory pathway. Then, based on the HK-2 cell model of hypothermic OGD, we found that CIRP silencing significantly stimulated the expression of the TGF-β1/p38 MAPK inflammatory pathway by activating the PHD3/HIF-1α axis, and induced more severe apoptosis through the mitochondrial cytochrome c-Apaf-1-caspase 9 and FADD-caspase 8 death receptor pathways compared with untransfected cells. However, silencing PHD3 remarkably activated the expression of HIF-1α and alleviated the apoptosis of HK-2 cells in hypothermic OGD. On this basis, by pretreating HK-2 and rats with enarodustat, a novel HIF-1α stabilizer, we found that enarodustat significantly mitigated renal cellular apoptosis under hypothermic IR and reversed the aggravated IRI induced by CIRP defect, both in vitro and in vivo.ConclusionOur findings indicated that CIRP may confer renoprotection against hypothermic IRI by suppressing PHD3/HIF-1α-mediated apoptosis. PHD3 inhibitors and HIF-1α stabilizers may have clinical value in renal IRI.

Cardiac Electrophysiological Changes and Downregulated Connexin 43 Prompts Reperfusion Arrhythmias Induced by Hypothermic Ischemia-Reperfusion Injury in Isolated Rat Hearts.

The purpose of this study was to determine the utility of the monophasic action potential (MAP) changes as an arrhythmic biomarker in hypothermic ischemia-reperfusion. The hypothermic ischemia-reperfusion model was subjected to 60min of cardioplegic arrest while the isolated rat hearts were preserved with a multidose cold K-H solution at 4°C. During the reperfusion period, the heart's arrhythmia and monophasic action potential were also monitored. The myocardial damage was assessed using HE and TTC stains. Immunohistochemistry and Western blotting were used to assess the expression and distribution of Connexin 43 (Cx43) and Akt. Collectively, prolonged action potential durations, increased dispersion of repolarization, and downregulated and lateralized Cx43 all contribute to the derangement of electrical impulse propagation that may underlie arrhythmogenesis in the cold ischemic heart following cardioplegic arrest. MAP might be used as a biomarker for arrhythmias caused by hypothermic ischemia-reperfusion.

Alveolar Epithelial Damage and Dysfunction as Common Features of Ischemia/Reperfusion Injury and Primary Graft Dysfunction in Lung Transplants

Purpose Ischemia reperfusion injury (I/R) is the major cause of primary graft dysfunction (PGD) after lung transplantation. I/R and PGD feature alveolar epithelial and endothelial damage, lung edema and inflammation. Edema resorption then depends on the restoration of alveolar epithelial integrity and the ability of alveolar cells to reabsorb Na+ (through ENaC channels) and fluid. We hypothesized that alveolar epithelial damage, and repair, are critical in PGD pathophysiology and resolution. Therefore, our aim was to identify novel biomarkers and therapeutic targets associated with I/R using cellular and animal models as well as human samples from lung transplants. Methods The impact of a protocol mimicking hypothermic ischemia and warm reperfusion was first tested on primary rat alveolar epithelial cell cultures. Then, the impact of I/R was studied in vivo using models of inflammatory stress induced by LPS infusion or after unilateral lung transplantation in pigs. Finally, lung biopsies from donor grafts were collected during lung transplantations. After defining PGD grades within the recipients, alveolar alterations were finally analyzed within donor graft tissues. Results In primary rat cell cultures, the I/R mimicking protocol impaired tight junction proteins (ZO-1), transepithelial resistance, wound repair capacity as well as expression in ENaC and CFTR channels. Treatment with a KvLQT1 K+ channel activator (R-L3) accelerated the repair rates and enhanced barrier integrity (ZO-1 staining) as well as ENaC and CFTR protein expressions. In the porcine models, an exacerbated inflammatory response was observed along with alveolar damage, lung edema and decreased ZO-1, ENaC and CFTR expressions. Our preliminary analyses also indicated that among lung transplants subsequently developing PGD, an inflammatory response and reduced ENaC, CFTR and ZO-1 expressions were already observed within biopsies from donor grafts, collected before transplantation. Conclusion Altogether our data support the hypothesis of alveolar epithelial dysfunction, after I/R injury. Based on these observations, our goal is to investigate a potential relationship between donor phenotypic factors, markers of epithelial damage/dysfunction within the grafts, inflammatory levels and the subsequent development of PGD in lung recipients.

Network construction of aberrantly expressed miRNAs and their target mRNAs in ventricular myocardium with ischemia\u2013reperfusion arrhythmias

BackgroundHypothermic ischemia-reperfusion arrhythmia remains the main factor affecting cardiac resuscitation under cardiopulmonary bypass. Existing research shows that certain miRNAs exhibit significantly different expressions and effects in arrhythmias, however, the effect of miRNAs on the progression of hypothermic ischemic–reperfusion arrhythmias (RA) and its potential mechanism remain to be further explored.MethodsSprague-Dawley (SD) rats were randomly divided into two groups (n = 8): a normal control group (Group C) and a hypothermic ischemia-reperfusion group (Group IR), which were used to establish a Langendorff isolated cardiac perfusion model. According to the arrhythmia scoring system, rats in group IR were divided into a high-risk group (IR-H) and a low-risk group (IR-L). miRNAs expression profiles of ventricular myocardium with global hypothermic ischemia–reperfusion and those of ventricular myocardium with hypothermic ischemia–RA were established through high-throughput sequencing. Furthermore, the aberrantly expressed miRNAs in myocardium with and without hypothermic ischemia–RA were screened and verified. The target genes of these aberrantly expressed miRNAs were predicted using RNAhybrid and MiRanda software. Based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we determined the mRNA targets associated with these miRNAs and studied the miRNA–mRNA interaction during the cardiovascular disease progression. The aberrantly expressed miRNAs related to hypothermic ischemia–RA were validated by Real-time Quantitative polymerase chain reaction (RT-qPCR).ResultsEight significantly aberrantly expressed miRNAs (rno-miR-122-5p, rno-miR-429, novel_miR-1, novel_miR-16, novel_miR-17, novel_miR-19, novel_miR-30, and novel_miR-43) were identified, among which six were up-regulated and two were down-regulated. Moreover, target genes and signaling pathways associated with these aberrantly expressed miRNAs were predicted and analyzed. The miRNA–mRNA interaction network graph showed that GJA1 gene was considered as the target of novel_miR-17.ConclusionsAberrantly expressed miRNAs were possibly associated with the formation mechanism of hypothermic ischemia–RA. Specific miRNAs, such as novel_miR-17 and rno-miR-429 are probably new potential targets for further functional studies of hypothermic ischemia–RA.

Prolonged duration of repolarization and decreased conduction velocity in the atrial myocardium after hypothermic ischemia-reperfusion may be related to expressions of inward rectifier potassium channel 2.1 protein and connexin 40.

The study aimed to determine the role of inward rectifier potassium channel 2.1 protein and connexin 40 expressions in regulating the duration of repolarization and conduction velocity of right atrial myocardium in rats following hypothermic ischemia-reperfusion. The Langendorff isolated rat cardiac perfusion models were divided into control (C) and hypothermic ischemia-reperfusion groups, with 8 models in group C and 16 models in group ischemia-reperfusion. Depending on the incidence of atrial arrhythmia after reperfusion, the models in group ischemia-reperfusion were further divided into reperfusion non-atrial arrhythmia or reperfusion atrial arrhythmia subgroup. Right atrial monophasic action potential duration at 50% and 90% of repolarization after 30 minutes of continuous perfusion in group C and group ischemia-reperfusion (T0), 105 minutes of continuous perfusion in group C or after 15 minutes of reperfusion in group ischemia-reperfusion (T1) and 120 minutes of continuous perfusion in group C or 30 minutes of reperfusion in group ischemia-reperfusion (T2) were recorded. Right atrial conduction velocity and effective refractory period were recorded at T2. Then, the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 in the right atrial myocardium were detected. Monophasic action potential duration at 50% and 90% were higher at T1 and T2 than those at T0 in subgroup reperfusion atrial arrhythmia (p < 0.05); monophasic action potential duration at 50% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); monophasic action potential duration at 90% in subgroup reperfusion atrial arrhythmia were larger than group C and subgroup reperfusion non-atrial arrhythmia at T1 and T2 (p < 0.05); effective refractory period in subgroup reperfusion atrial arrhythmia was greater than that in group C and subgroup reperfusion non-atrial arrhythmia, and the conduction velocity and the expressions of inward rectifier potassium channel 2.1 protein and connexin 40 were significantly lower than group C and subgroup reperfusion non-atrial arrhythmia (p < 0.05). The prolonged duration of repolarization and a decrease in conduction velocity of the atrial myocardium occur in rats after hypothermic ischemia-reperfusion. These observed effects may be related to the downregulated expressions of connexin 40 and inward rectifier potassium channel 2.1.

Electrophysiological changes of atrial myocardium in a rat model of hypothermic ischemia-reperfusion: an in vitro experiment

Objective To evaluate the electrophysiological changes of atrial myocardium in a rat model of hypothermic ischemia-reperfusion (I/R). Methods Sixteen isolated Sprague-Dawley rat hearts successfully perfused in the Langendorff apparatus were divided into control group (group C) and hypothermic I/R group (group IR) using a random number table method, with 8 heats in each group.Heats in group IR were further divided into reperfusion-non-atrial arrhythmia subgroup (group R-NAA) and reperfusion-atrial arrhythmia subgroup (group R-AA) depending on whether atrial arrhythmia occurred after reperfusion.In group C, the heart was perfused with K-H solution at 37 ℃ for 120 min.In group IR, the heart was perfused with K-H solution at 37 ℃ for 30 min and then perfusion was stopped, cardiac arrest was induced for 60 min through injecting Thomas solution (4 ℃, 20 ml/kg), the area around the heart was protected with low temperature (4 ℃) Thomas solution, and hearts were resuscitated with 4 ℃ Thomas solution (10 ml/kg) at 30 min after cardiac arrest and with 37 ℃ K-H solution for 30 min staring from 60 min after cardiac arrest.At 30 min of equilibration (T0), 105 min of equilibration/15 min of reperfusion (T1), and 120 min of equilibration/30 min of reperfusion (T2), right atrial monophasic action potentials, maximal velocity of phase zero, monophasic action potential amplitude (MAPA) and MAP duration at 50% and 90% of repolarization (MAPD50 and MAPD90) were measured.Right-atrium conduction velocity and effective refractory period were recorded at T2, and the ratio of ERP to MAPD90 (ERP/MAPD90) was calculated.Atrial fibrillation was induced by programmed electrical stimulation, and the maximum pacing cycle length of inducing atrial fibrillation (AF-PCLmax) was recorded. Results Compared with C and R-NAA groups, the maximal velocity of phase zero was significantly decreased and MAPD90 was increased at T1, the right-atrium conduction velocity and ERP/MAPD90 ratio were decreased and MAPD90, effective refractory period and AF-PCLmax were increased at T2 in group R-AA (P<0.05). Conclusion The decrease in depolarization velocity, prolongation of repolarization duration and decrease in conduction velocity, excitability and electrical stability may be the electrophysiological mechanism of reperfused atrial arrhythmia in rats. Key words: Myocardial reperfusion injury; Arrhythmias, cardiac; Heart atria

Prolonged myocardial protection during hypothermic storage: potential application for cardiac surgery and myocardial tissue engineering

The increasing need for donor organs for heart transplantation and promising progress in myocardial tissue engineering requires more effective long-term storage strategies to provide adequate recipient supply and accelerate translation of novel strategies for myocardial regeneration and reconstruction. We designed a multi-segmental model to analyze the capability of different solutions to preserve cardiomyocytes, contractile engineered 3-dimensional myocardial tissue and whole hearts at hypothermia. Four chemically defined hypothermic storage media and two clinical cardioplegic solutions were compared in standardized hypothermia-reperfusion protocols. We identified an animal-free and chemically defined solution (ChillProtec), not yet in clinical use, that attenuated hypothermic ischemia-reperfusion injury of single cardiomyocytes and multicellular myocardial tissue constructs significantly. Stored for up to five days in hypothermic conditions (4 °C–8 °C), cardiomyocytes showed significantly less cell death and maintained physiological function, i.e. contractility and force development, which was in part prevented by cell swelling and inhibition of apopotosis. In an ex vivo-model mimicking clinical situations, we were able to preserve adequate function in Langendorff-perfused isolated rat hearts after hypothermic storage. Our findings have direct implications on both in vitro and pre-clinical in vivo studies on novel regenerative strategies involving cardiomyocytes, such as drug development and reconstructive cell therapies. Moreover, the prolonged biopreservation described here may serve as basis to further optimize hypothermia-associated routines for cardiac surgery concerning intra-operative cardioplegic arrest or organ preservation for transport and extended storage.

ERK5 promotes TLR2-dependent up-regulation of inflammatory mediator expression induces by global myocardial ischemia

Global myocardial ischemia and reperfusion [I/R] injury is predominantly associated with heart transplant and coronary artery bypass graft (CABG) surgery. Toll-like receptor-2 (TLR2) is a pivotal pathogen recognition receptor that has a key role in inflammation and injury. The proinflammatory role for ERK5 downstream of TLR2 in global myocardial I/R has not been reported previously. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by ERK5 of TLR2. Global myocardial I/R was induced in male TLR2+/+ and TLR2−/− mice using heterotopic abdominal heart transplantation model that allows to subject donor hearts to global hypothermic ischemia and blood reperfusion, which very closely stimulates I/R conditions associated with cardiac surgical operations. The results show that inhibition or siRNA knockdown of ERK5 reduces the TLR2-dependent activation of expression of cytokines, attenuated myocardial injury, less oxidative stress and decreased NF-κB activation and indicating that ERK5 promotes cytokine expression downstream of TLR2. We have identified novel aspects of TLR2 signaling in cold myocardial I/R, including that ERK5 activity is required for TLR2 activation.

The effects of profound hypothermia on pancreas ischemic injury: a new experimental model.

Pancreatic ischemia-reperfusion (IR) has a key role in pancreas surgery and transplantation. Most experimental models evaluate the normothermic phase of the IR. We proposed a hypothermic model of pancreas IR to evaluate the benefic effects of the cold ischemic phase. We performed a reproducible model of hypothermic pancreatic IR. The ischemia was induced in the pancreatic tail portion (1-hour ischemia, 4-hour reperfusion) in 36 Wistar rats. They are divided in 3 groups as follows: group 1 (control), sham; group 2, normothermic IR; and group 3, hypothermic IR. In group 3, the temperature was maintained as close to 4.5°C. After reperfusion, serum amylase and lipase levels, inflammatory mediators (tumor necrosis factor α, interleukin 6), and pancreas histology were evaluated. In pancreatic IR groups, amylase, cytokines, and histological damage were significantly increased when compared with group 1. In the group 3, we observed a significant decrease in tumor necrosis factor α (P = 0.004) and interleukin 6 (P = 0.001) when compared with group 2. We did not observe significant difference in amylase (P = 0.867), lipase (P = 0.993), and histology (P = 0.201). In our experimental model, we reproduced the cold phase of pancreas IR, and the pancreas hypothermia reduced the inflammatory mediators after reperfusion.

Ghrelin reduces myocardial injury following global ischemia and reperfusion via suppression of myocardial inflammatory response

Ghrelin is a small endogenous peptide principally produced and secreted by the gastric mucosa, with a major role in appetite and metabolism regulation. We hypothesized that anti-inflammatory therapy, as produced by exogenous administration of ghrelin, would decrease the myocardial inflammatory response to global hypothermia I/R, thereby affording myocardial protection. Heterotopic cervical heart transplantation that allows to subject donor hearts to global hypothermic ischemia and blood reperfusion, which very closely stimulates I/R conditions associated with cardiac surgical operations. Ghrelin administration prior to blood reperfusion significantly decreased serum concentrations of cTn-I versus animals subjected I/R alone, with a significantly attenuated VCAM-1 expression in I/R animals pre-treated with ghrelin. The tissue concentrations of pro-inflammatory cytokines (IL-6, IL-1β, and MCP-1) were ameliorated by the administration of ghrelin prior to reperfusion versus the concentrations observed in animals subjected to I/R alone. Significantly fewer monocytes in the tissue sections of I/R+ghrelin animals versus those subjected to I/R alone. Exogenous ghrelin administration prior to reperfusion of an ischemic heart resulted in a significant reduction in myocardial injury as measured by cTn-I. The reduced myocardial injury was accompanied by an attenuated tissue expression of several pro-inflammatory mediators, including VCAM-1, IL-6, IL-1β, and MCP-1.

Catalytic peroxynitrite decomposition improves reperfusion injury after heart transplantation

Peroxynitrite, a reactive nitrogen species, has been implicated in the development of ischemia-reperfusion injury. The present study investigated the effects of the potent peroxynitrite decomposition catalyst FP15 on myocardial and endothelial function after hypothermic ischemia-reperfusion in a heterotopic rat heart transplantation model. After a 1-hour ischemic preservation and implantation of donor hearts, reperfusion was started after application of vehicle (5% glucose solution) or FP15 (0.3 mg/kg). The assessment of left ventricular pressure-volume relations, total coronary blood flow, endothelial function, immunohistochemical markers of nitro-oxidative stress, and myocardial high-energy phosphates was performed at 1 and 24 hours of reperfusion. After 1 hour of reperfusion, myocardial contractility (maximal slope of systolic pressure increment at 140 μL left ventricular volume: 5435 ± 508 mm Hg/s vs 2346 ± 263 mm Hg/s), coronary blood flow (3.98 ± 0.33 mL/min/g vs 2.74 ± 0.29 mL/min/g), and endothelial function were significantly improved, nitro-oxidative stress was reduced, and myocardial high-energy phosphate content was preserved in the FP15-treated animals compared with controls. Pharmacologic peroxynitrite decomposition reduces reperfusion injury after heart transplantation as the result of reduction of nitro-oxidative stress and prevention of energy depletion and exerts a beneficial effect against reperfusion-induced graft cardiac and coronary endothelial dysfunction.

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function.

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3β, and ERK1/2. There was also maintenance of cardiac myocyte β-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.

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.

Granulocyte Colony-Stimulating Factor Prevents Reperfusion Injury After Heart Preservation

Heart transplantation is an accepted method of treatment for selected patients with end-stage heart disease. Making prolonged heart preservation safer will benefit patients awaiting heart transplantation. Granulocyte colony-stimulating factor (G-CSF) exhibited protective effects against myocardial ischemia-reperfusion injury mediated through the Janus kinase (Jak)/(signal transducer and activator of transcription (Stat) pathway. We examined whether pharmacologic preconditioning with G-CSF improves cardiac function after heart preservation. Male rats were divided into four groups: group A, saline injection; group B, G-CSF, 10 microg/kg; group C, G-CSF, 100 microg/kg; and group D, G-CSF, 100 microg/kg plus AG490 (a selective Jak2 inhibitor), 1 mg/kg. The G-CSF and AG490 were given intravenously for 3 consecutive days. Four hours after the final treatment, isolated rat hearts underwent 12 hours of hypothermic (4 degrees C) preservation, followed by 60 minutes of normothermic reperfusion. Stat3 phosphorylation was observed in the heart at 15 minutes after G-CSF treatment in group C, but this was attenuated by additional treatment with AG 490 in group D. Compared with group A, group C exhibited significant recovery of left ventricular pressure, maximum positive rate of left ventricular developed pressure (Max dP/dt), and coronary flow (p < 0.05, respectively), as well as lower creatine phosphokinase leakage during reperfusion (p < 0.05). Group B and group D did not show significant hemodynamic recovery during reperfusion. In group C, increased Bcl-xL and decreased Bax expressions as well as decreased terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL)-positive cardiomyocytes were observed after reperfusion. Immunohistochemical examination showed significantly increased capillary density before hypothermic preservation in group C, but not in other groups. Pharmacologic preconditioning with G-CSF protected hearts from prolonged hypothermic ischemia-reperfusion injury.

Poly(ADP-ribose) polymerase and renal hypothermic preservation injury.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) has been implicated in ischemia-reperfusion injury in many tissues under normothermic conditions. The purpose of this study was to determine whether PARP contributes to mechanisms of the hypothermic ischemia-reperfusion injury that occurs when kidneys are cold stored for transplantation. Cortical tissue slice PARP enzyme activity rose significantly with prolonged cold storage and was dependent on both reperfusion and preservation quality. However, prior exposure to warm ischemia abrogated this increase. PARP protein increased with cold storage but was not dependent on reperfusion. PARP enzyme activity rose quickly after reperfusion in buffer and was not different when whole blood was used. Addition of exogenous hydrogen peroxide (3 mM) to normal renal slices significantly increased PARP activity over 4 h in the cortex but not in the medulla, but the medullary basal PARP synthesis rate was five times higher than that in the cortex. However, the reactive oxygen species (ROS) inhibitors catalase (2,000 U/ml), Trolox (200 microM), and DMSO (15 mM) did not reduce reperfusion-induced PARP activity in cold-stored cortical slices. Finally, PARP inhibitors potentiated preservation injury in isolated canine proximal renal tubules. In conclusion, canine renal PARP enzyme activity rises with prolonged cold storage after reperfusion and may play a protective rather than an injurious role in hypothermic preservation for transplantation. ROS are sufficient but not necessary to activate PARP under these conditions.

Effects of inhibition of myocardial extracellular-responsive kinase and P38 mitogen-activated protein kinase on mechanical function of rat hearts after prolonged hypothermic ischemia.

Mitogen-activated protein kinases (MAPKs), including extracellular-responsive kinase (ERK) and p38 MAPK, are activated by stresses associated with hypothermia-rewarming and ischemia-reperfusion. Their activation in heart is associated with beneficial (preconditioning) and adverse effects (apoptosis and impaired contractility). This study determined whether ERK and p38 MAPK activities are altered by hypothermic ischemia and normothermic reperfusion and the consequences of their inhibition on recovery of myocardial function. Left ventricular work (L x min(-1) x mm Hg) was assessed during normothermic perfusion (30 min) of isolated rat hearts that were either freshly excised or previously subjected to hypothermic storage (8 hr, 3 degrees C) and rewarming (10 min, 37 degrees C) before normothermic reperfusion (30 min). Phospho-specific immunoblot analysis of p38 MAPK was performed in hearts and various cultured cells. Compared with fresh hearts, hearts subjected to hypothermia and rewarming demonstrated impaired left ventricular work (1.96+/-0.53, n=12 vs. 8.37+/-0.46, n=4, <0.05) during reperfusion. The ERK inhibitor, PD98059 (20 microM), present during storage and rewarming, caused modest improvement (3.66+/-0.75, n=9, <0.05). The p38 MAPK inhibitor, SB202190 (10 microM), when present during reperfusion, improved recovery (to 6.12+/-0.75, n=6, <0.05); it was ineffective if present only during rewarming (1.52+/-0.88, n=4). In rat2 fibroblasts, hypothermia and rewarming activated p38 MAPK and its downstream kinase MAPK-activated protein kinase 2, but not c-Jun N-terminal kinase/stress-activated protein kinase. Myocardial p38 MAPK and MAPK-activated protein kinase 2 are stimulated by hypothermia, ischemia, and rewarming and are detrimental to recovery of mechanical function of hearts subjected to prolonged hypothermic storage. Inhibition of p38 MAPK may be useful in protocols to improve the recovery of mechanical function of cold-stored hearts.

37 Comparative study between normo and hypothermic ischemia-reperfusion liver injury in a rat model: Preliminary results

37 Comparative study between normo and hypothermic ischemia-reperfusion liver injury in a rat model: Preliminary results

Increase in Content of Glycogen Phosphorylase during Ischemia in Isolated Rabbit Heart.

The amount of a 97-kDa protein in the sarcoplasmic reticulum-enriched microsomal fraction from rabbit heart changed in a manner dependent on the oxygenated condition after global hypothermic ischemia and subsequent normothermic reperfusion. Isolated hearts were immersed in physiologic saline for 0h (control group) or 1, 2 and 3h at 4°C (ischemia group) and were subjected to 40-min reperfusion at 37°C after 1h of hypothermic ischemia (reperfused group). The amount of the 97-kDa protein in the microsomal fraction apparently increased after ischemia, but decreased after reperfusion. The N-terminal amino acid of the protein was blocked and two internal sequences were determined, demonstrating the 97-kDa protein to be glycogen phosphorylase. A polyclonal antibody against rabbit skeletal muscular glycogen phosphor-ylase (GP) stained only the 97-kDa protein in the microsomal fraction in Western blot analysis. Microsomal fractions isolated from rabbit brain, liver, spleen, and white skeletal muscle were also examined by Western blot analysis using anti-GP. Liver and skeletal muscle contained a 97-kDa protein that reacted with anti-GP in control preparations. Ischemia induced a modest increase in the amount of the 97-kDa protein skeletal muscle microsomes, whereas no change in the amount of the protein occurred in liver microsomes. The results indicate that alteration in the energy metabolism from ischemia to the subsequent reperfusion is associated with the amount of GP.

Arachidonic Acid Metabolism in Intestinal Hypothermic Preservation Injury

The effect of hypothermic intestinal ischemia and short-term reperfusion on mucosal arachidonic acid metabolism was studied in a dog model of intestinal preservation injury. Canine intestinal segments were flushed with cold Collins solution, cold stored (4°C) for either 24 or 48 h, and subsequently reperfused in the donor for 1 h. Samples of intestinal mucosa obtained before ischemia, after the ischemia period, and after the reperfusion period were placed into tissue culture, and arachidonic acid metabolites were measured in the tissue incubation media. Prostaglandin E2(PGE2) and prostacyclin (PGI2) production significantly increased after 24 h of cold ischemia and after 1 h of reperfusion, respectively. Intestines cold stored for 48 h and after 1 h of reperfusion produced significantly elevated quantities of thromboxane B2, PGI2, PGE2, and leukotriene B4, relative to the production rates from nonischemic control tissue or tissue subjected to 48 h of hypothermic ischemia without reperfusion. Mucosal production of thiol ether leukotrienes (LTC4, LTD4, LTE4) was not altered by ischemia or reperfusion at any time of cold ischemia. The synthesis of the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) was not altered by hypothermic ischemia or reperfusion, but this arachidonate metabolite was produced by small intestinal mucosa in the greatest quantities. Specifically, nanogram quantities of 12-HETE were produced by intestinal mucosa compared to picogram quantities of the other metabolites measured. Significant synthesis of the delta lactone derivative of 5-hydroxyeicosatetraenoic acid was detected by HPLC in many tissue samples undergoing 48 h of ischemia and reperfusion, relative to nonischemic tissue samples. In conclusion, significant increases in arachidonate cyclooxygenase and lipoxygenase metabolites have been identified in intestinal mucosa subjected to long-term hypothermic ischemia and short-term reperfusion. Synthesis of these products increases with the duration of cold ischemia and may play a role in intestinal preservation injury.

Protective effects of N-acetylcysteine on hypothermic ischemia-reperfusion injury of rat liver

We investigated whether intraportal injection of 150 mg/kg N-acetylcysteine (NAC) into rats reduced hepatic ischemia-reperfusion injury after 48 hours of cold storage and 2 hours of reperfusion. The organ was isolated and perfused to evaluate liver function. The control group received an intraportal injection of 5% dextrose. NAC increased L-cysteine concentrations 15 minutes after injection (1.29 +/- 0.11 mumol/g vs. 2.68 +/- 0.4 mumol/g, P < .05). However, neither treatment modified glutathione liver concentrations relative to preinjection values. After 48 hours of cold storage and 2 hours of reperfusion, livers from NAC-treated rats produced larger amounts of bile than those in the control group (5.04 +/- 1.92 vs. 0.72 +/- 0.37 microL/g liver; P < .05), and showed a significant reduction in liver injury, as indicated by reduced release of lactate dehydrogenase (679.4 +/- 174.4 vs. 1891.3 +/- 268.3 IU/L/g; P < .01), aspartate transaminase (AST) (13.94 +/- 3.5 vs. 38.75 IU/L/g; P < .01), alanine transaminase ALT) (14.92 +/- 4.09 vs. 45.91 +/- 10.58 IU/L/g; P < .05), and acid phosphatase, a marker of Kupffer cell injury (344.4 +/- 89.6 vs. 927.3 +/- 150.8 IU/L/g; P < .01) in the perfusate. Reduced glutathione concentrations in the perfusate were similar in the two groups (805 +/- 69 vs. 798 +/- 252 nmol/L/g), whereas oxidized glutathione (GSSG) concentrations were higher in the control group (967 +/- 137 vs. 525 +/- 126 nmol/L/g; P < .05). Reduced glutathione (GSH) concentrations in liver tissue collected at the end of perfusion were significantly higher in the NAC group (7.3 +/- 0.9 vs. 4.1 +/- 1.0 mumol/g; P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)