Warm Reperfusion Research Articles

Exercise Preconditioning of the Donor Liver Decreases Cold Ischemia/Reperfusion Injury in a Mouse Model.

Liver transplantation stands as the primary treatment for end-stage liver disease, with demand surging in recent decades because of expanded indications. However, hepatic ischemia/reperfusion injury can lead to liver transplant failure in both deceased donor and living donor transplantation. This study explored whether preconditioning donor livers through exercise training (ExT) could mitigate cold ischemic injury posttransplantation. Donor C57BL/6 mice underwent ExT via treadmill running or remained sedentary. After 4 wk, the donor liver underwent cold storage and subsequent orthotopic liver transplantation or ex vivo warm reperfusion. Donor liver from mice subjected to ExT showed significantly decreased hepatic injury on reperfusion. Tissue histology revealed decreased sinusoidal congestion, vacuolization, and hepatocellular necrosis in livers from ExT mice, and immunofluorescence staining further revealed a decreased number of apoptotic cells in ExT grafts. Livers from ExT donors expressed decreased intragraft inflammatory cytokines cascade, decreased neutrophil infiltration and neutrophil extracellular traps, and increased M2 phenotype of recipient macrophages compared with grafts from sedentary mice. After cold storage, liver grafts from ExT donors showed decreased accumulation of reactive oxygen species and decreased levels of cytochrome c and high mobility group box 1 released in the liver effluent. In addition, ExT grafts showed upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and higher levels of mitochondrial content. Similar effects of decreased hepatic injury were observed in wild-type mice when pretreated with a PGC-1α stimulator ZLN005 instead of ExT. These findings suggest that augmenting hepatocytic mitochondrial content through donor exercise or PGC-1α stimulation may offer therapeutic avenues to mitigate postreperfusion inflammation and improve transplant outcomes.

Assessment of temporal changes in glomerular filtration rate following clamp ischemia in male and female rats using transcutaneous FITC-sinistrin clearance

Warm arterial clamping with reperfusion in rats is a commonly utilized model of ischemic kidney injury (IRI). Traditionally, changes in plasma creatinine or creatinine clearance have been utilized to assess kidney function in this model. A limitation of these approaches, however, is that changes in plasma creatinine are relatively slow to occur, resulting in low temporal fidelity. This limits our ability to relate changes in kidney function to histo-pathological changes observed during reperfusion of the kidneys. Transcutaneous assessment of FITC-sinistrin clearance enables accurate, repeated measurement of glomerular filtration rate in conscious animals over short segments of time (hours) without drawing blood. The goal of the current study was to characterize temporal changes in renal function in male and female rats after reperfusion from bi-lateral warm arterial clamp ischemia using transcutaneous FITC-sinistrin clearance. Our study was performed in age matched male (n=8) and female (n=5) Sprague Dawley rats (12-20wks of age). FITC sinistrin clearance was measured by affxing a miniaturized transcutaneous measurement device between the shoulder blades (MediBeacon) of rats. 50mg/kg of FITC-sinistrin was then injected i.v via the tail vein and the device allowed to record for ~3 hours. Measurements were taken either at pre-surgery (baseline), 1-3 hours, 24 hours or 7 days following reperfusion of the kidneys from warm bilateral arterial clamp ischemia. For the ischemic surgery, animals were anesthetized with isoflurane (2-5%, 95% O2) and the right and left kidneys were accessed by flank incisions. After being exposed, the renal arteries were separated from the renal veins via dissection. Both renal arteries were then clamped with microserrefines for 45 minutes. After the ischemic period, the clamps were removed, allowing blood flow to be restored to the kidneys. Body temperature was measured via a rectal probe and maintained at 37°C throughout the procedure by a servo-controlled heating table and UV heating lamp. The wounds were then closed, and the animals allowed to recover. Buprenorphine was administered as an analgesic. For each measurement, the ½ life of FITC sinistrin was determined using a 1-compartment model (MB Lab software) and glomerular filtration rate (GFR) per 100g of body weight rate calculated. Baseline GFR was 0.94±0.03ml/min/100g in males and 1.09±0.02/min/100g in females (PTTEST=0.04). Following 45 minutes of warm bilateral arterial ischemia and reperfusion, GFR fell to 0.24±0.08ml/min/100g in male and 0.36±0.15ml/min/100g in females within 1-3 hours of reperfusion. At 24 hours of reperfusion, GFR averaged 0.06±0.03ml/min/100g in males and 0.28±0.16ml/min/100g in females. GFR recovered in males and females by 7 days of reperfusion averaging 0.86±0.10ml/min/100g in males and 0.83±0.00ml/min/100g in females. There was no significant difference between FITC-sinistrin clearance between males and females at any time point after IRI. Our data indicate that most of the reduction in GFR is present immediately after releasing the clamp. This is consistent with continued vascular disturbances mediating the initial decline in glomerular filtration rate in this model (i.e. vasospasm). The absence of sex differences in GFR decline using this method suggests that sex-differences detected using plasma creatinine may be, at least in part, secondary to the more rapid production of creatinine in males than in females or greater baseline filtration/body weight ratio in females compared to males rather than differences in renal function decline to ischemia. American Heart Association Transformational Project Award 970585 to Paul O'Connor. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury

BackgroundCellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia–reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes.MethodsSCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs).ResultsAfter 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression.ConclusionAdding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

Comprehensive bile acid pool analysis during ex-vivo liver perfusion in a porcine model of ischemia-reperfusion injury

Bile acids (BA) are key for liver regeneration and injury. This study aims at analyzing the changes in the BA pool induced by ischemia-reperfusion (IRI) and investigates the impact of hypothermic oxygenated perfusion (HOPE) on the BA pool compared to static cold storage (SCS). In a porcine model of IRI, liver grafts underwent 30 min of asystolic warm ischemia followed by 6 h of SCS (n = 6) ± 2 h of HOPE (n = 6) and 2 h of ex-situ warm reperfusion. The BA pool in bile samples was analyzed with liquid chromatography coupled with tandem mass spectrometry. We identified 16 BA and observed significant changes in response to ischemia-reperfusion, which were associated with both protective and injury mechanisms. Second, HOPE-treated liver grafts exhibited a more protective BA phenotype, characterized by a more hydrophilic BA pool compared to SCS. Key BA, such as GlycoCholic Acid, were identified and were associated with a decreased transaminase release and improved lactate clearance during reperfusion. Partial Least Square-Discriminant Analysis revealed a distinct injury profile for the HOPE group. In conclusion, the BA pool changes with liver graft IRI, and preservation with HOPE results in a protective BA phenotype compared to SCS.

A Single Preservation Solution for Static Cold Storage and Hypothermic Oxygenated Perfusion of Marginal Liver Grafts: A Preclinical Study.

Hypothermic oxygenated perfusion (HOPE) improves outcomes of marginal liver grafts. However, to date, no preservation solution exists for both static cold storage (SCS) and HOPE. After 30 min of asystolic warm ischemia, porcine livers underwent 6 h of SCS followed by 2 h of HOPE. Liver grafts were either preserved with a single preservation solution (IGL2) designed for SCS and HOPE (IGL2-Machine Perfusion Solution [MPS] group, n = 6) or with the gold-standard University of Wisconsin designed for for SCS and Belzer MPS designed for HOPE (MPS group, n = 5). All liver grafts underwent warm reperfusion with whole autologous blood for 2 h, and surrogate markers of hepatic ischemia-reperfusion injury (IRI) were assessed in the hepatocyte, cholangiocyte, vascular, and immunological compartments. After 2 h of warm reperfusion, livers in the IGL2-MPS group showed no significant differences in transaminase release (aspartate aminotransferase: 65.58 versus 104.9 UI/L/100 g liver; P = 0.178), lactate clearance, and histological IRI compared with livers in the MPS group. There were no significant differences in biliary acid composition, bile production, and histological biliary IRI. Mitochondrial and endothelial damage was also not significantly different and resulted in similar hepatic inflammasome activation. This preclinical study shows that a novel IGL2 allows for the safe preservation of marginal liver grafts with SCS and HOPE. Hepatic IRI was comparable with the current gold standard of combining 2 different preservation solutions (University of Wisconsin + Belzer MPS). These data pave the way for a phase I first-in-human study and it is a first step toward tailored preservation solutions for machine perfusion of liver grafts.

Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells

BackgroundIschemia–reperfusion injury is a key complication following lung transplantation. The clinical application of ex vivo lung perfusion (EVLP) to assess donor lung function has significantly increased the utilization of “marginal” donor lungs with good clinical outcomes. The potential of EVLP on improving organ quality and ameliorating ischemia–reperfusion injury has been suggested.MethodsTo determine the effects of ischemia–reperfusion and EVLP on gene expression in human pulmonary microvascular endothelial cells and epithelial cells, cell culture models were used to simulate cold ischemia (4 °C for 18 h) followed by either warm reperfusion (DMEM + 10% FBS) or EVLP (acellular Steen solution) at 37 °C for 4 h. RNA samples were extracted for bulk RNA sequencing, and data were analyzed for significant differentially expressed genes and pathways.ResultsEndothelial and epithelial cells showed significant changes in gene expressions after ischemia–reperfusion or EVLP. Ischemia–reperfusion models of both cell types showed upregulated pro-inflammatory and downregulated cell metabolism pathways. EVLP models, on the other hand, exhibited downregulation of cell metabolism, without any inflammatory signals.ConclusionThe commonly used acellular EVLP perfusate, Steen solution, silenced the activation of pro-inflammatory signaling in both human lung endothelial and epithelial cells, potentially through the lack of serum components. This finding could establish the basic groundwork of studying the benefits of EVLP perfusate as seen from current clinical practice.

Tackling regulated cell death yields enhanced protection in lung grafts.

Background: Effective preservation strategies to ameliorate lung graft ischaemia injury are needed to rescue 'extended criteria' or 'marginal' lung grafts, and to improve recipient outcomes after transplantation. Methods: Lung grafts from male Lewis rats were extracted after 40 min of cardiocirculatory death, and healthy human lung tissues were collected from patients undergoing a lobectomy. Lung samples were then preserved in a 4°C preservation solution supplemented with 0.1 nM Dexmedetomidine (Dex, α2-adrenoceptor agonist) for 16 h. In vitro, human lung epithelial A549 cells were preserved in the 4°C preservation solution with 0.1 nM Dex for 24 h, then re-cultured in the cell culture medium at 37°C to mimic the clinical scenario of cold ischaemia and warm reperfusion. Lung tissues and cells were then analysed with various techniques including western blot, immunostaining and electron microscope, to determine injuries and the protection of Dex. Results: Prolonged warm ischaemia after cardiocirculatory death initiated Rip kinase-mediated necroptosis, which was exacerbated by cold storage insult and enhanced lung graft injury. Dex supplementation significantly reduced necroptosis through upregulating Nrf2 activation and reducing oxidative stress, thereby significantly improving lung graft morphology. Dex treatment also attenuated endoplasmic reticulum stress, stabilised lysosomes and promoted cell membrane resealing function, consequently reducing cell death and inflammatory activation after hypothermic hypoxia-reoxygenation in A549 cells. Conclusions: Inhibition of regulated cell death through Dex supplementation to the graft preservation solution improves allograft quality which may aid to expand the donor lung pool and enhance lung transplant outcomes per se.

Effects of iNOS in Hepatic Warm Ischaemia and Reperfusion Models in Mice and Rats: A Systematic Review and Meta-Analysis.

Warm ischaemia is usually induced by the Pringle manoeuver (PM) during hepatectomy. Currently, there is no widely accepted standard protocol to minimise ischaemia-related injury, so reducing ischaemia-reperfusion damage is an active area of research. This systematic review and meta-analysis focused on inducible nitric oxide synthase (iNOS) as an early inflammatory response to hepatic ischaemia reperfusion injury (HIRI) in mouse- and rat-liver models. A systematic search of studies was performed within three databases. Studies meeting the inclusion criteria were subjected to qualitative and quantitative synthesis of results. We performed a meta-analysis of studies grouped by different HIRI models and ischaemia times. Additionally, we investigated a possible correlation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) regulation with iNOS expression. Of 124 included studies, 49 were eligible for the meta-analysis, revealing that iNOS was upregulated in almost all HIRIs. We were able to show an increase of iNOS regardless of ischemia or reperfusion time. Additionally, we found no direct associations of eNOS or NO with iNOS. A sex gap of primarily male experimental animals used was observed, leading to a higher risk of outcomes not being translatable to humans of all sexes.

Treprostinil Supplementation Ameliorates Hepatic Ischemia Reperfusion Injury and Regulates Expression of Hepatic Drug Transporters: An Isolated Perfused Rat Liver (IPRL) Study

PurposeIR injury is an unavoidable consequence in deceased donor liver transplantation. Cold preservation and warm reperfusion may change the expression and function of drug transporters in the liver due to vasoconstriction, infiltration of neutrophils and release of cytokines. We hypothesize that vasodilation, anti-platelet aggregation and proinflammatory downregulation activities of treprostinil will diminish the IR injury and its associated effects.MethodsLivers obtained from male SD rats (n = 20) were divided into 1) Control, 2) IR, 3) Treprostinil-1 (preservation only), and 4) Treprostinil-2 (preservation and reperfusion) groups. Control livers were procured and immediately reperfused. Livers in the other groups underwent preservation for 24 h and were reperfused. All the livers were perfused using an Isolated Perfused Rat Liver (IPRL) system. Periodic perfusate, cumulative bile samples and liver tissue at the end of perfusion were collected. Liver injury markers, bile flow rates, m-RNA levels for uptake and efflux transporters (qRT-PCR) were measured.ResultsCold preservation and warm reperfusion significantly increased the release of AST and ALT in untreated livers. Treprostinil supplementation substantially reduced liver injury. Bile flow rate was significantly improved in treprostinil-2 group. m-RNA levels of Slc10a1, Slc22a1, and Slc22a7 in liver were increased and m-RNA levels of Mdr1a were decreased by IR. Treprostinil treatment increased Abcb11 and Abcg2 m-RNA levels and maintained Slc22a1m-RNA similar to control livers.ConclusionsTreprostinil treatment significantly reduced liver injury. IR injury changed expression of both uptake and efflux transporters in rat livers. Treprostinil significantly altered the IR injury mediated changes in m-RNA expression of transporters.

Prolyl Hydroxylase Inhibition Mitigates Allograft Injury During Liver Transplantation.

Ischemia and reperfusion injury (IRI) determines primary allograft function after liver transplantation (LT). Primary graft dysfunction (PGD) is associated with increased morbidity and impaired graft survival and can eventually progress to graft failure requiring retransplantation. Hypoxia-inducible transcription factor-prolyl hydroxylase containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors, which control the adaptive hypoxia response through the hypoxia-inducible factor (HIF). In this study, we have investigated pharmacological activation of the HIF pathway through inhibition of PHDs as a strategy to reduce PGD after LT. Primary rat hepatocytes were isolated and the impact of the pan-PHD small-molecule inhibitor ethyl-3,4-dihydroxybenzoate (EDHB) on HIF-1 and its downstream target gene expression assessed. Subsequently, various rodent models of segmental warm liver ischemia and reperfusion and orthotopic LT were applied to study the impact of EDHB on normothermic or combined cold and warm liver IRI. Liver enzyme levels and histology were analyzed to quantify hepatic IRI. In vitro, EDHB induced HIF-1 signaling and significantly upregulated its downstream target heme-oxygenase 1 in primary rat hepatocytes. In vivo, after establishment of the optimal EDHB pretreatment conditions in a murine IRI model, EDHB pretreatment significantly mitigated hepatic IRI after warm segmental liver ischemia and reperfusion and allograft injury after orthotopic LT in rats. Mechanistically, EDHB stabilized HIF-1 in the liver and subsequently increased hepatoprotective heme-oxygenase 1 levels, which correlated with reduced hepatic IRI in these models. This proof-of-concept study establishes a strong therapeutic rationale for targeting PHDs with small-molecule inhibitors to mitigate PGD after LT.

Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts

Background: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. Methods: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 °C (controlled oxygenated rewarming—COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. Results: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 °C was effectuated in the same controlled manner. Conclusion: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

Augmented Cardiac Mitochondrial Capacity in High Capacity Aerobic Running "Disease-Resistant" Phenotype at Rest Is Lost Following Ischemia Reperfusion.

Rationale: Regular active exercise is considered therapeutic for cardiovascular disease, in part by increasing mitochondrial respiratory capacity, but a significant amount of exercise capacity is determined genetically. Animal models, demonstrating either high capacity aerobic running (HCR) or low capacity aerobic running (LCR) phenotypes, have been developed to study the intrinsic contribution, with HCR rats subsequently characterized as “disease resistant” and the LCRs as “disease prone.” Enhanced cardioprotection in HCRs has been variable and mutifactoral, but likely includes a metabolic component. These studies were conducted to determine the influence of intrinsic aerobic phenotype on cardiac mitochondrial function before and after ischemia and reperfusion.Methods: A total of 34 HCR and LCR rats were obtained from the parent colony at the University of Toledo, housed under sedentary conditions, and fed normal chow. LCR and HCR animals were randomly assigned to either control or ischemia-reperfusion (IR). On each study day, one HCR/LCR pair was anesthetized, and hearts were rapidly excised. In IR animals, the hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to global hypothermic ischemic arrest (80 min). Following the arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff perfusion system. Following reperfusion, the heart was weighed and the left ventricle (LV) was isolated. A midventricular ring was obtained to estimate infarction size [triphenyltetrazolium chloride (TTC)] and part of the remaining tissue (~150 mg) was transferred to a homogenation buffer on ice. Isolated mitochondria (MITO) samples were prepared and used to determine respiratory capacity under different metabolic conditions. In control animals, MITO were obtained and prepared similarly immediately following anesthesia and heart removal, but without IR.Results: In the control rats, both resting and maximally stimulated respiratory rates were higher (32 and 40%, respectively; p < 0.05) in HCR mitochondria compared to LCR. After IR, resting MITO respiratory rates were decreased to about 10% of control in both strains, and the augmented capacity in HCRs was absent. Maximally stimulated rates also were decreased more than 50% from control and were no longer different between phenotypes. Ca++ retention capacity and infarct size were not significantly different between HCR and LCR (49.2 ± 5.6 vs. 53.7 ± 4.9%), nor was average coronary flow during reperfusion or arrhythmogenesis. There was a significant loss of mitochondria following IR, which was coupled with decreased function in the remaining mitochondria in both strains.Conclusion: Cardiac mitochondrial capacity from HCR was significantly higher than LCR in the controls under each condition. After IR insult, the cardiac mitochondrial respiratory rates were similar between phenotypes, as was Ca++ retention capacity, infarct size, and arrhythmogenicity, despite the increased mitochondrial capacity in the HCRs before ischemia. Relatively, the loss of respiratory capacity was actually greater in HCR than LCR. These data could suggest limits in the extent to which the HCR phenotype might be “protective” against acute tissue stressors. The extent to which any of these deficits could be “rescued” by adding an active exercise component to the intrinsic phenotype is unknown.

Ischemia-Reperfusion Injury in a Simulated Lung Transplant Setting Differentially Regulates Transcriptomic Profiles between Human Lung Endothelial and Epithelial Cells.

Current understanding of mechanisms of ischemia-reperfusion-induced lung injury during lung preservation and transplantation is mainly based on clinical observations and animal studies. Herein, we used cell and systems biology approaches to explore these mechanisms at transcriptomics levels, especially by focusing on the differences between human lung endothelial and epithelial cells, which are crucial for maintaining essential lung structure and function. Human pulmonary microvascular endothelial cells and human lung epithelial cells were cultured to confluent, subjected to different cold ischemic times (CIT) to mimic static cold storage with preservation solution, and then subjected to warm reperfusion with a serum containing culture medium to simulate lung transplantation. Cell morphology, viability, and transcriptomic profiles were studied. Ischemia-reperfusion injury induced a CIT time-dependent cell death, which was associated with dramatic changes in gene expression. Under normal control conditions, endothelial cells showed gene clusters enriched in the vascular process and inflammation, while epithelial cells showed gene clusters enriched in protein biosynthesis and metabolism. CIT 6 h alone or after reperfusion had little effect on these phenotypic characteristics. After CIT 18 h, protein-biosynthesis-related gene clusters disappeared in epithelial cells; after reperfusion, metabolism-related gene clusters in epithelial cells and multiple gene clusters in the endothelial cells also disappeared. Human pulmonary endothelial and epithelial cells have distinct phenotypic transcriptomic signatures. Severe cellular injury reduces these gene expression signatures in a cell-type-dependent manner. Therapeutics that preserve these transcriptomic signatures may represent new treatment to prevent acute lung injury during lung transplantation.

Regulation of autophagy protects against liver injury in liver surgery-induced ischaemia/reperfusion.

Transient ischaemia and reperfusion in liver tissue induce hepatic ischaemia/reperfusion (I/R) tissue injury and a profound inflammatory response in vivo. Hepatic I/R can be classified into warm I/R and cold I/R and is characterized by three main types of cell death, apoptosis, necrosis and autophagy, in rodents or patients following I/R. Warm I/R is observed in patients or animal models undergoing liver resection, haemorrhagic shock, trauma, cardiac arrest or hepatic sinusoidal obstruction syndrome when vascular occlusion inhibits normal blood perfusion in liver tissue. Cold I/R is a condition that affects only patients who have undergone liver transplantation (LT) and is caused by donated liver graft preservation in a hypothermic environment prior to entering a warm reperfusion phase. Under stress conditions, autophagy plays a critical role in promoting cell survival and maintaining liver homeostasis by generating new adenosine triphosphate (ATP) and organelle components after the degradation of macromolecules and organelles in liver tissue. This role of autophagy may contribute to the protection of hepatic I/R‐induced liver injury; however, a considerable amount of evidence has shown that autophagy inhibition also protects against hepatic I/R injury by inhibiting autophagic cell death under specific circumstances. In this review, we comprehensively discuss current strategies and underlying mechanisms of autophagy regulation that alleviates I/R injury after liver resection and LT. Directed autophagy regulation can maintain liver homeostasis and improve liver function in individuals undergoing warm or cold I/R. In this way, autophagy regulation can contribute to improving the prognosis of patients undergoing liver resection or LT.

Effects of graft preservation conditions on coronary endothelium and cardiac functional recovery in a rat model of donation after circulatory death

Use of cardiac grafts obtained with donation after circulatory death (DCD) could significantly improve donor heart availability. As DCD hearts undergo potentially deleterious warm ischemia and reperfusion, clinical protocols require optimization to ensure graft quality. Thus, we investigated effects of alternative preservation conditions on endothelial and/or vascular and contractile function in comparison with the current clinical standard. Using a rat DCD model, we compared currently used graft preservation conditions, St. Thomas n°2 (St. T) at 4°C, with potentially more suitable conditions for DCD hearts, adenosine-lidocaine preservation solution (A-L) at 4°C or 22°C. Following general anesthesia and diaphragm transection, hearts underwent either 0 or 18 min of in-situ warm ischemia, were explanted, flushed and stored for 15 min with either St. T at 4°C or A-L at 4°C or 22°C, and then reperfused under normothermic, aerobic conditions. Endothelial integrity and contractile function were determined. Compared to 4°C preservation, 22°C A-L significantly increased endothelial nitric oxide synthase (eNOS) dimerization and reduced oxidative tissue damage (p < 0.05 for all). Furthermore, A-L at 22°C better preserved the endothelial glycocalyx and coronary flow compared with St. T, tended to reduce tissue calcium overload, and stimulated pro-survival signaling. No significant differences were observed in cardiac function among ischemic groups. Twenty-two-degree Celsius A-L solution better preserves the coronary endothelium compared to 4°C St. T, which likely results from greater eNOS dimerization, reduced oxidative stress, and activation of the reperfusion injury salvage kinase (RISK) pathway. Improving heart preservation conditions immediately following warm ischemia constitutes a promising approach for the optimization of clinical protocols in DCD heart transplantation.

Dual Versus Single Oxygenated Hypothermic Machine Perfusion of Porcine Livers: Impact on Hepatobiliary and Endothelial Cell Injury.

Hypothermic oxygenated machine perfusion (HOPE) reduces ischemia-reperfusion injury of donor livers and is increasingly used in clinical transplantation. However, it remains unclear whether perfusion via the portal vein alone (HOPE) or via both the portal vein and hepatic artery (dual HOPE or DHOPE) is superior. Twelve porcine livers donated after circulatory death were randomized for 2 h of HOPE (n = 6) or DHOPE (n = 6), followed by 4 h of warm reperfusion with whole blood, to mimic transplantation. Hepatobiliary and endothelial cell function and injury markers were determined in perfusate and bile samples. Biopsies of bile ducts, hepatic arteries, and liver parenchyma were collected to assess histological damage and the expression of endothelial protective genes (KLF-2, eNOS, ET-1, CD31, VWF, VEGF-A). There were no differences in hepatobiliary function and injury after warm reperfusion between the groups, apart from a 2-fold lower concentration of alanine aminotransferase in the perfusate (P = 0.045) and a lower peak lactate dehydrogenase in bile (P = 0.04) of livers preserved by DHOPE. Endothelial cell function and injury, as assessed by perfusate nitric oxide and von Willebrand factor antigen levels, as well as endothelial protective gene expressions, were similar between the groups. The hepatic arteries of both groups showed no microscopic evidence of injury. This study did not reveal major differences in hepatobiliary or endothelial function and injury after preservation by single or dual HOPE of porcine livers donated after circulatory death.

Hyperkalemia: Major but still understudied complication among heart transplant recipients.

Hyperkalemia is a recognized and potentially life-threatening complication of heart transplantation. In the complex biosystem created by transplantation, recipients are susceptible to multiple mechanisms for hyperkalemia which are discussed in detail in this manuscript. Hyperkalemia in heart transplantation could occur pre-transplant, during the transplant period, or post-transplant. Pre-transplant causes of hyperkalemia include hypothermia, donor heart preservation solutions, conventional cardioplegia, normokalemic cardioplegia, continuous warm reperfusion technique, and ex-vivo heart perfusion. Intra-transplant causes of hyperkalemia include anesthetic medications used during the procedure, heparinization, blood transfusions, and a low output state. Finally, post-transplant causes of hyperkalemia include hemostasis and drug-induced hyperkalemia. Hyperkalemia has been studied in kidney and liver transplant recipients, but there is limited data on the incidence, causes, management, and prevention in heart transplant recipients. Hyperkalemia is associated with an increased risk of hospital mortality and readmission in these patients. This review describes the current literature pertaining to the causes, pathophysiology, and treatment of hyperkalemia in patients undergoing heart transplantation and focuses primarily on post-heart transplantation.

Protective effects of molecular hydrogen on lung injury from lung transplantation.

Lung grafts may experience multiple injuries during lung transplantation, such as warm ischaemia, cold ischaemia, and reperfusion injury. These injuries all contribute to primary graft dysfunction, which is a major cause of morbidity and mortality after lung transplantation. As a potential selective antioxidant, hydrogen molecule (H2) protects against post-transplant complications in animal models of multiple organ transplantation. Herein, the authors review the current literature regarding the effects of H2 on lung injury from lung transplantation. The reviewed studies showed that H2 improved the outcomes of lung transplantation by decreasing oxidative stress and inflammation at the donor and recipient phases. H2 is primarily administered via inhalation, drinking hydrogen-rich water, hydrogen-rich saline injection, or a hydrogen-rich water bath. H2 favorably modulates signal transduction and gene expression, resulting in the suppression of pro-inflammatory cytokines and excess reactive oxygen species production. Although H2 appears to be a physiological regulatory molecule with antioxidant, anti-inflammatory and anti-apoptotic properties, its exact mechanisms of action remain elusive. Taken together, accumulating experimental evidence indicates that H2 can significantly alleviate transplantation-related lung injury, mainly via inhibition of inflammatory cytokine secretion and reduction in oxidative stress through several underlying mechanisms. Further animal experiments and preliminary human clinical trials will lay the foundation for the use of H2 as a treatment in the clinic.

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.

Effects of Graft Preservation Conditions on Coronary Endothelium and Functional Recovery in a Rat Model of Donation after Circulatory Death

Purpose Donation after circulatory death (DCD) could significantly improve cardiac graft availability. However, DCD hearts are subjected to potentially damaging conditions prior to circulatory arrest and during warm ischemia and reperfusion. Although the feasibility of DCD heart transplantation has been demonstrated, protocols for graft preservation and storage require optimization to ensure graft quality. Therefore, we aimed to compare the current clinical conditions used for heart preservation (St. Thomas n°2) with a potentially more suitable solution for DCD hearts, evaluating endothelial and cardiac function to investigate differences in cardioprotective effects. Methods Following anaesthesia, diaphragm transection, and circulatory arrest in male Wistar rats to simulate DCD conditions, hearts were subjected to either 0 or 18 minutes of warm, in-situ ischemia. At end-ischemia, hearts were perfused with either: St. Thomas n°2 cardioplegia at 4°C as in current clinical practice, or normokalemic, adenosine-lidocaine cardioplegia (A-L) at 4°C or 22°C. Afterwards, hearts were stored for 15 min in their respective solutions, and then reperfused ex-situ for either 30 min (to assess endothelial function) or 60 min (to assess cardiac function and biochemical parameters). Results A-L cardioplegia at 22°C better preserves vascular function as compared to cold A-L or St. T cardioplegia, as demonstrated by greater eNOS coupling (p Conclusion Use of A-L and avoidance of profound hypothermia and hyperkalemic cardioplegia could improve endothelial and contractile function, potentially through the promotion of eNOS dimerization and activation of the RISK pathway; these approaches should help to optimize clinical protocols in DCD heart transplantation and facilitate its widespread adoption in clinical practice.