Optimizing resuscitation of the donation after circulatory death heart by mitochondrial protection in a female porcine model.

BACKGROUND: Due to the shortage of donor organs, an increasing number of transplant organs are harvested after circulatory arrest (donation after circulatory death, DCD). Using a translational porcine DCD model, we developed and evaluated a protocol based on cardioprotection by multi-drug postconditioning to optimize resuscitation of DCD hearts by ex situ heart perfusion (ESHP). METHODS: Hearts of female pigs (45.0±4.5 kg) were procured following a clinically identical DCD protocol, consisting of the termination of ventilator support and confirmation of circulatory arrest, followed by a 15-min standoff period. DCD hearts were randomly allocated to ESHP (38.4°C) in the absence (untreated, N=5) or presence (treated, N=5) of a postconditioning treatment added to the perfusate, consisting of Intralipid (1%), sevoflurane (2% v/v), and remifentanil (3 nM). All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 60 min in Langendorff mode and for additional 300 min in working mode for a total perfusion time of 6 hrs. Oxidative capacity and detailed left ventricular (LV) mechanical function under increasing workload (left atrial pressure 6-12 mmHg) were assessed hourly. LV tissue was snap-frozen at the end of ESHP and used for molecular analyses. RESULTS: LV inotropy (LVdP/dtmax) did not decline over time in treated DCD hearts and was significantly higher at the end of the protocol as compared with untreated DCD hearts (ΔLVdP/dtmax= 440 mmHg/s, p=0.009). Treated DCD hearts exhibited persistently higher LV stroke work index (LVSWI) during the 6-hr period of ESHP, whereas untreated DCD hearts displayed a significant decline (ΔLVSWI=-3.10 mL*mmHg/g, p(time within untreated group)<0.001). Treated DCD hearts displayed higher metabolic activity as measured by oxygen consumption (ΔO2=3.11 mL O2/min/100g; p=0.004), and released lower amounts of cell-free mitochondrial DNA into the perfusate, a marker of potential graft dysfunction. Treated hearts also used fatty acids from Intralipid as an energy source, while untreated DCD hearts showed glyceroneogenesis with triglyceride accumulation and depletion of tricarboxylic acid cycle intermediates, reduced mitochondrial complex I, II, and III activities with accumulation of mitochondrial NADH, and signs of ultrastructural damage. CONCLUSIONS: A translationally relevant protective ESHP protocol consisting of treatment with Intralipid, sevoflurane, and remifentanil markedly accelerated functional recovery and improved viability of DCD hearts.

The Effects of Oxygen-Derived Free-Radical Scavengers During Normothermic Ex-Situ Heart Perfusion.

Oxidative stress occurs during ex-situ heart perfusion (ESHP) and may negatively affect functional preservation of the heart. We sought to assess the status of key antioxidant enzymes during ESHP, and the effects of augmenting these antioxidants on the attenuation of oxidative stress and improvement of myocardial and endothelial preservation in ESHP. Porcine hearts were perfused for 6 hours with oxygen-derived free-radical scavengers polyethylene glycol (PEG)-catalase or PEG-superoxide dismutase (SOD) or with naive perfusate (control). The oxidative stress-related modifications were determined in the myocardium and coronary vasculature, and contractile function, injury, and endothelial integrity were compared between the groups. The activity of key antioxidant enzymes decreased and adding catalase and SOD restored the enzyme activity. Cardiac function and endothelial integrity were preserved better with restored catalase activity. Catalase and SOD both decreased myocardial injury and catalase reduced ROS production and oxidative modification of proteins in the myocardium and coronary vasculature. The activity of antioxidant enzymes decrease in ESHP. Catalase may improve the preservation of cardiac function and endothelial integrity during ESHP. While catalase and SOD may both exert cardioprotective effects, unbalanced SOD and catalase activity may paradoxically increase the production of reactive species during ESHP.

Cyclosporine A Does Not Mitigate Liver Ischemia/Reperfusion Injury in an Ex Vivo Porcine Model of Donation After Circulatory Death.

BACKGROUND Ischemia/reperfusion injury (IRI) is an inherent problem in organ transplantation, owing to the obligate period of ischemia that organs must endure. Cyclosporine A (CsA), though better know as an immunosuppressant, has been shown to mitigate warm IRI in a variety of organ types, including the liver. However, there is little evidence for CsA in preventing hepatic IRI in the transplant setting. MATERIAL AND METHODS In the present study, we tested the effect of CsA on hepatic IRI in a large-animal ex vivo model of donation after circulatory death (DCD). Porcine donors were pre-treated with either normal saline control or 20 mg/kg of CsA. Animals were subject to either 45 or 60 minutes of warm ischemia before hepatectomy, followed by 2 or 4 hours of cold storage prior to reperfusion on an ex vivo circuit. Over the course of a 12-hour perfusion, perfusion parameters were recorded and perfusate samples and biopsies were taken at regular intervals. RESULTS Peak perfusate lactate dehydrogenase was significantly decreased in the lower-ischemia group treated with CsA compared to the untreated group (4220 U/L [3515-5815] vs 11 305 [10 100-11 674]; P=0.023). However, no difference was seen between controls and CsA-treated groups on other parameters in perfusate alanine or asparagine aminotransferase (P=0.912, 0.455, respectively). Correspondingly, we found no difference on midpoint histological injury score (P=0.271). CONCLUSIONS We found minimal evidence that CsA is protective against hepatic IRI in our DCD model.

Oxidative stress and related metabolic alterations are induced in ex situ perfusion of donated hearts regardless of the ventricular load or leukocyte depletion.

We sought to determine the role of donor blood circulating leukocytes in mediating oxidative stress and inflammation during normothermic ex situ heart perfusion (ESHP). Normothermic ESHP allows preservation of donated heart in a perfused, dynamic state, preventing ischemia. However, the cardiac function declines during ESHP, limiting the potential of this method for improvement of the outcomes of transplantation and expanding the donor pool. Extracorporeal circulation-related oxidative stress plays a critical role in the functional decline of the donor heart. Hearts from domestic pigs were perfused in working mode (WM, whole blood-based or leukocyte-depleted blood-based perfusate) or nonworking mode. Markers of oxidative stress and responsive glucose anabolic pathways were induced in the myocardium regardless of left ventricular load. Myocardial function during ESHP as well as cardioprotective mechanisms were preserved better in WM. Leukocyte-depleted perfusate did not attenuate tissue oxidative stress or perfusate proinflammatory cytokines and did not improve functional preservation. Although ESHP is associated with ongoing oxidative stress and metabolic alteration in the myocardium, preserved cardioprotective mechanisms in WM may exert beneficial effects. Leukocyte depletion of the perfusate may not attenuate inflammation and oxidative stress effectively or improve the functional preservation of the heart during ESHP.

Model of Acute Liver Failure in an Isolated Perfused Porcine Liver-Challenges and Lessons Learned.

Acute liver failure (ALF) is a rare but devastating disease associated with substantial morbidity and a mortality rate of almost 45%. Medical treatments, apart from supportive care, are limited and liver transplantation may be the only rescue option. Large animal models, which most closely represent human disease, can be logistically and technically cumbersome, expensive and pose ethical challenges. The development of isolated organ perfusion technologies, originally intended for preservation before transplantation, offers a new platform for experimental models of liver disease, such as ALF. In this study, female domestic swine underwent hepatectomy, followed by perfusion of the isolated liver on a normothermic machine perfusion device. Five control livers were perfused for 24 h at 37 °C, while receiving supplemental oxygen and nutrition. Six livers received toxic doses of acetaminophen given over 12 h, titrated to methemoglobin levels. Perfusate was sampled every 4 h for measurement of biochemical markers of injury (e.g., aspartate aminotransferase [AST], alanine aminotransferase [ALT]). Liver biopsies were taken at the beginning, middle, and end of perfusion for histological assessment. Acetaminophen-treated livers received a median dose of 8.93 g (8.21-9.75 g) of acetaminophen, achieving a peak acetaminophen level of 3780 µmol/L (3189-3913 µmol/L). Peak values of ALT (76 vs. 105 U/L; p = 0.429) and AST (3576 vs. 4712 U/L; p = 0.429) were not significantly different between groups. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p = 0.016). All acetaminophen treated livers developed significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p = 0.004). The development of a model of ALF in the ex vivo setting was confounded by the development of toxic methemoglobinemia. Further attempts using alternative agents or dosing strategies may be warranted to explore this setting as a model of liver disease.

The evaluation of constant coronary artery flow versus constant coronary perfusion pressure during normothermic ex situ heart perfusion.

BACKGROUND: Evidence suggests that hearts that are perfused under ex-situ conditions lose normal coronary vasomotor tone and experience contractile failure over a few hours. We aimed to evaluate the effect of different coronary perfusion strategies during ex situ heart perfusion on cardiac function and coronary vascular tone. METHODS: Porcine hearts (n = 6 each group) were perfused in working mode for 6 hours with either constant aortic diastolic pressure (40 mmHg) or constant coronary flow rate (500 mL/min). Functional and metabolic parameters, cytokine profiles, cardiac and vascular injury, coronary artery function and oxidative stress were compared between groups. RESULTS: Constant coronary flow perfusion demonstrated better functional preservation and less edema formation (Cardiac index: flow control = 8.33 vs pressure control = 6.46 mL·min-1·g-1, p = 0.016; edema formation: 7.92% vs 19.80%, p < 0.0001). Pro-inflammatory cytokines, platelet activation as well as endothelial activation were lower in the flow control group. Similarly, less cardiac and endothelial injury was observed in the constant coronary flow group. Evaluation of coronary artery function showed there was loss of coronary autoregulation in both groups. Oxidative stress was induced in the coronary arteries and was relatively lower in the flow control group. CONCLUSIONS: A strategy of controlled coronary flow during ex situ heart perfusion provides superior functional preservation and less edema formation, together with less myocardial damage, leukocyte, platelet, endothelial activation, and oxidative stress. There was loss of coronary autoregulation and decrease of coronary vascular resistance during ESHP irrespective of coronary flow control strategy. Inflammation and oxidative stress state in the coronary vasculature may play a role.

Inflammation and Oxidative Stress in the Context of Extracorporeal Cardiac and Pulmonary Support.

Extracorporeal circulation (ECC) systems, including cardiopulmonary bypass, and extracorporeal membrane oxygenation have been an irreplaceable part of the cardiothoracic surgeries, and treatment of critically ill patients with respiratory and/or cardiac failure for more than half a century. During the recent decades, the concept of extracorporeal circulation has been extended to isolated machine perfusion of the donor organ including thoracic organs (ex-situ organ perfusion, ESOP) as a method for dynamic, semi-physiologic preservation, and potential improvement of the donor organs. The extracorporeal life support systems (ECLS) have been lifesaving and facilitating complex cardiothoracic surgeries, and the ESOP technology has the potential to increase the number of the transplantable donor organs, and to improve the outcomes of transplantation. However, these artificial circulation systems in general have been associated with activation of the inflammatory and oxidative stress responses in patients and/or in the exposed tissues and organs. The activation of these responses can negatively affect patient outcomes in ECLS, and may as well jeopardize the reliability of the organ viability assessment, and the outcomes of thoracic organ preservation and transplantation in ESOP. Both ECLS and ESOP consist of artificial circuit materials and components, which play a key role in the induction of these responses. However, while ECLS can lead to systemic inflammatory and oxidative stress responses negatively affecting various organs/systems of the body, in ESOP, the absence of the organs that play an important role in oxidant scavenging/antioxidative replenishment of the body, such as liver, may make the perfused organ more susceptible to inflammation and oxidative stress during extracorporeal circulation. In the present manuscript, we will review the activation of the inflammatory and oxidative stress responses during ECLP and ESOP, mechanisms involved, clinical implications, and the interventions for attenuating these responses in ECC.

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP.

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the in vivo validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

The Position of the Heart During Normothermic Ex Situ Heart Perfusion is an Important Factor in Preservation and Recovery of Myocardial Function.

Ex situ heart perfusion (ESHP) is being investigated as a method for the continuous preservation of the myocardium in a semiphysiologic state for subsequent transplantation. Most methods of ESHP position the isolated heart in a hanging (H) state, representing a considerable departure from the in vivo anatomical positioning of the heart and may negatively affect the functional preservation of the heart. In the current study, cardiac functional and metabolic parameters were assessed in healthy pig hearts, perfused for 12 hours, in either an H, or supported (S) position, either in nonworking mode (NWM) or working mode (WM). The cardiac function was best preserved in the S position hearts in WM (median 11 hour cardiac index (CI)/1 hour CI%: working mode perfusion in supported position = 94.77% versus nonworking mode perfusion in supported position = 62.80%, working mode perfusion in H position = 36.18%, nonworking mode perfusion in H position = 9.75%; p < 0.001). Delivery of pyruvate bolus significantly improved the function in S groups, however, only partially reversed myocardial dysfunction in the H heart groups. The hearts perfused ex situ in a semianatomical S position and in physiologic WM had better functional preservation and recovery than the H hearts in non-S position. Optimizing the positional support for the ex situ-perfused hearts may improve myocardial preservation during ESHP.

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A Low-Cost Perfusate Alternative for Ex Vivo Lung Perfusion.

BACKGROUND: Normothermic ex vivo lung perfusion (EVLP) has been used successfully to evaluate and recondition marginal donor lungs; however, multiple barriers continue to prevent its widespread adoption. We sought to develop a common hospital ingredient-derived perfusate (CHIP) with equivalent functional and inflammatory characteristics to a standard Krebs-Henseleit buffer with 8% serum albumin-derived perfusate (KHB-Alb) to improve access and reduce costs of ex vivo organ perfusion. METHODS: Sixteen porcine lungs were perfused using negative pressure ventilation (NPV) EVLP for 12 hours in a normothermic state and were allocated equally to 2 groups: KHB-Alb vs CHIP. Physiological parameters, cytokine profiles, and edema formation were compared between treatment groups. RESULTS: Perfused lungs in both groups demonstrated equivalent oxygenation (partial pressure of arterial oxygen/fraction of inspired oxygen ratio >350 mm Hg) and physiological parameters. There was equivalent generation of tumor necrosis factor-α and IL-6, irrespective of perfusate solution used, when comparing CHIP vs KHB-Alb. Pig lungs developed equivalent edema formation between groups (CHIP: 15.8 ± 4.8%, KHB-Alb 19.5 ± 4.4%, P > .05). CONCLUSION: A perfusate derived of common hospital ingredients provides equivalent results to a standard Krebs-Henseleit buffer with 8% serum albumin-based perfusate in NPV-EVLP.

Immunity and Stress Responses Are Induced During Ex Situ Heart Perfusion.

BACKGROUND: Ex situ heart perfusion (ESHP) preserves the donated heart in a perfused, beating condition preventing cold storage-related ischemia and provides a platform to evaluate myocardial viability during preservation. However, myocardial function declines gradually during ESHP. Extracorporeal circulation systems are associated with the induction of systemic inflammatory and stress responses. Our aim was to evaluate the incidence of inflammation and induction of endoplasmic reticulum stress responses during an extended period of ESHP. METHODS: Cardiac function, myocardial tissue injury, markers of inflammation, oxidative stress, and endoplasmic reticulum stress were assessed in healthy pig hearts, perfused for 12 hours either in nonworking mode (non-WM=7) or working mode (WM, n=6). RESULTS: Cardiac function declined during ESHP but was significantly better preserved in the hearts perfused in WM (median 11-hour cardiac index/1-hour cardiac index: WM=27% versus non-WM=9.5%, P=0.022). Myocardial markers of endoplasmic reticulum stress were expressed higher in ESHP hearts compared with in vivo samples. The proinflammatory cytokines and oxidized low-density lipoprotein significantly increased in the perfusate throughout the perfusion in both perfusion groups. The left ventricular expression of the cytokines and malondialdehyde was induced in non-WM, whereas it was not different between WM and in vivo. CONCLUSIONS: Myocardial function declines during ESHP regardless of perfusion mode. However, ESHP in WM may lead to superior preservation of myocardial function and viability. Both inflammation and endoplasmic reticulum stress responses are significantly induced during ESHP and may contribute to the myocardial functional decline, representing a potential therapeutic target to improve the clinical donor heart preservation.

Trends in the Explanatory or Pragmatic Nature of Cardiovascular Clinical Trials Over 2 Decades.

Importance: Pragmatic trials test interventions using designs that produce results that may be more applicable to the population in which the intervention will be eventually applied. Objective: To investigate how pragmatic or explanatory cardiovascular (CV) randomized clinical trials (RCT) are, and if this has changed over time. Data Source: Six major medical and CV journals, including New England Journal of Medicine, Lancet, JAMA, Circulation, European Heart Journal, and Journal of the American College of Cardiology. Study Selection: All CV-related RCTs published during 2000, 2005, 2010, and 2015 were identified and included. Data Extraction and Synthesis: Included RCTs were assessed by 2 independent adjudicators with expertise in RCT and CV medicine. Main Outcomes and Measures: The outcome measure was the level of pragmatism evaluated using the Pragmatic Explanatory Continuum Index Summary (PRECIS)-2 tool, which uses a 5-point ordinal scale (ranging from very pragmatic to very explanatory) across 9 domains of trial design, including eligibility, recruitment, setting, organization, intervention delivery, intervention adherence, follow-up, primary outcome, and analysis. Results: Of 616 RCTs, the mean (SD) PRECIS-2 score was 3.26 (0.70). The level of pragmatism increased over time from a mean (SD) score of 3.07 (0.74) in 2000 to 3.46 (0.67) in 2015 (P < .001 for trend; Cohen d relative effect size, 0.56). The increase occurred mainly in the domains of eligibility, setting, intervention delivery, and primary end point. PRECIS-2 score was higher for neutral trials than those with positive results (P < .001) and in phase III/IV trials compared with phase I/II trials (P < .001) but similar between different sources of funding (public, industry, or both; P = .38). More pragmatic trials had more sites, larger sample sizes, longer follow-ups, and mortality as the primary end point. Conclusions and Relevance: The level of pragmatism increased moderately over 2 decades of CV trials. Understanding the domains of current and future clinical trials will aid in the design and delivery of CV trials with broader application.

Total parenteral nutrition in ex vivo lung perfusion: Addressing metabolism improves both inflammation and oxygenation.

Ex vivo lung perfusion (EVLP) protocols generally limit metabolic supplementation to insulin and glucose. We sought to determine whether the addition of total parenteral nutrition (TPN) would improve lung function in EVLP. Ten porcine lungs were perfused using EVLP for 24 hours and supplemented with insulin and glucose. In the treatment group (n = 5), the perfusate was also supplemented with a continuous infusion of TPN containing lipids, amino acids, essential vitamins, and cofactors. Physiologic parameters and perfusate electrolytes were continuously evaluated. Perfusate lactate, lipid and branch chain amino acid (BCAA) concentrations were also analyzed to elucidate how substrates were being utilized over time. Lungs in the TPN group exhibited significantly better oxygenation. Perfusate sodium was more stable in the TPN group. In the control group, free fatty acids (FFA) were quickly depleted, reaching negligible levels early in the perfusion. Alternatively, BCAA in the control group rose continually over the perfusion demonstrating a shift toward proteolysis for energy substrate. In the TPN group, both FFA and BCAA concentrations remained stable at in vivo levels after initial stabilization. TNF-α concentrations were lower in the TPN group. The addition of TPN in EVLP allows for better electrolyte composition, decreased inflammation, and improved graft performance.

Continuous Hemodialysis Does Not Improve Graft Function During Ex Vivo Lung Perfusion Over 24 Hours.

BACKGROUND: Extended periods of ex vivo lung perfusion (EVLP) lead to several inadvertent consequences including accumulation of lactate and increasing electrolyte concentrations in the perfusate. We sought to determine whether continuous hemodialysis (CHD) of the perfusate would be a suitable modality for improving ionic homeostasis in extended EVLP without compromising functional outcomes. METHODS: Twelve porcine lungs were perfused using EVLP for 24 hours. All lungs were ventilated with negative pressure ventilation. Lungs in the treatment group (n = 6) underwent continuous hemodialysis of the perfusate. Functional parameters, edema formation, and histopathologic analysis were used to assess graft function. Electrolyte and lactate profiles were also followed to assess the efficiency of hemodialysis. RESULTS: Lungs in both treatment and control groups demonstrated stable and acceptable oxygenation to 24 hours. Lungs demonstrated a decrease in compliance over time. There was no difference in oxygenation and compliance between groups. CHD-EVLP lungs had higher pulmonary vascular resistance and pulmonary artery pressures. Despite increased perfusion pressures, weight gain at both 11 and 23 hours was not different between groups. Perfusate sodium and lactate concentrations were significantly lower in the CHD-EVLP group. CONCLUSION: The addition of continuous hemodialysis to EVLP did not improve graft function up to 24 hours despite improved maintenance of perfusate composition.

Clearance of transaminases during normothermic ex situ liver perfusion.

BACKGROUND: One of the most promising applications of liver normothermic machine perfusion (NMP) is the potential to directly assess graft viability and injury. In most NMP studies, perfusate transaminases are utilized as markers of graft injury. Our aim was to further elucidate the metabolism of transaminases by healthy porcine livers during NMP, specifically whether such livers could clear circuit perfusate transaminases. METHODS: A highly concentrated transaminase solution was prepared from homogenized liver, with an aspartate aminotransferase (AST) level of 107,427 U/L. Three livers in the treatment group were compared to three controls, during 48 hours of NMP. In the treatment group, the circuit perfusate was injected with the transaminase solution to artificially raise the AST level to a target of 7,500 U/L. Perfusate samples were taken at two-hour intervals and analyzed for biochemistry until NMP end. Graft oxygen consumption and vascular parameters were monitored. RESULTS: Compared to controls, treated perfusions demonstrated abrupt elevations in transaminase levels (p>0.0001) and lactate dehydrogenase (LDH) (p>0.0001), which decreased over time, but never to control baseline. Liver function, as demonstrated by lactate clearance and oxygen consumption was not different between groups. The treatment group demonstrated a higher portal vein resistance (p = 0.0003), however hepatic artery resistance was similar. Treated livers had higher bile production overall (p<0.0001). CONCLUSIONS: Addition of high levels of transaminases and LDH to a healthy porcine liver during ex situ perfusion results in progressive clearance of these enzymes, suggesting preserved liver metabolism. Such tolerance tests may provide valuable indicators of prospective graft function.

Myocardial Functional Decline During Prolonged Ex Situ Heart Perfusion.

BACKGROUND: Myocardial function declines in a time-dependent fashion during ex situ heart perfusion. Cell death and metabolic alterations may contribute to this phenomenon, limiting the safe perfusion period and the potential of ex situ heart perfusion to expand the donor pool. Our aim was to investigate the etiology of myocardial functional decline in ex situ perfused hearts. METHODS: Cardiac function, apoptosis, effectors and markers of cell death, and metabolic function were assessed in healthy pig hearts perfused for 12 hours. These hearts were perfused in nonworking mode or working mode. RESULTS: Cardiac function declined during ex situ heart perfusion regardless of perfusion mode but was significantly better preserved in the hearts perfused in working mode (11-hour cardiac index/1-hour cardiac index: working mode, 33%; nonworking mode, 10%; p = 0.025). The rate of apoptosis was higher in the ex situ perfused hearts compared with in vivo samples (apoptotic cells: in vivo, 0.13%; working mode, 0.54%; nonworking mode, 0.88%; p < 0.001), but the absolute values were low and out of proportion to the decline in function in either group. Myocardial dysfunction at the end of the perfusion interval was partially rescued by delivery of a pyruvate bolus. CONCLUSIONS: A significant decline in myocardial function occurs over time in hearts preserved ex situ that is out of proportion to the magnitude of myocyte cell death present in dysfunctional hearts. Alterations in myocardial substrate utilization during prolonged ex situ heart perfusion may contribute to this phenomenon and represent an avenue to improve donor heart preservation.

Enhanced myocardial protection in cardiac donation after circulatory death using Intralipid® postconditioning in a porcine model.

PURPOSE: Intralipid® (ILE), a clinically used lipid emulsion, reduces ischemia-reperfusion (IR) injury in healthy and infarct-remodelled rat hearts. We tested whether ILE is also cardioprotective in large porcine hearts in the context of the donation after circulatory death (DCD) model, where human hearts are procured for transplantation after cardiac arrest and thus are exposed to significant IR injury. METHODS: After induction of anesthesia, surgical preparation, termination of ventilator support, and cardiac arrest, hearts of female pigs were procured following a 15 min standoff period, with an optimized normokalemic crystalloid adenosine-lidocaine cardioplegia. Hearts were then randomly allocated to ex vivo reperfusion (38°C) in the absence (control) or presence of 1% ILE. All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 30 min in Langendorff mode and for an additional 30 min in working mode to assess mechanical function. Left ventricular (LV) biopsies were obtained after five minutes of reperfusion and LV tissue was preserved at the end of reperfusion for biochemical analyses and immunohistochemistry. RESULTS: Intralipid® postconditioning reduced cell membrane damage as assessed by the mean (standard deviation) leakage of myocardial glutathione disulfide (39 (9) nmol·mg-1 protein vs 19 (7) nmol·mg-1 protein; P = 0.006), protected LV tissue from protein carbonylation (3.4 [0.6] nmol·mg-1 protein vs 5.3 [0.9] nmol·mg-1 protein; P = 0.006), decreased myeloperoxidase activity (35 [8] nmol·min-1·mg-1 protein vs 75 [11] nmol·min-1·mg-1 protein; P < 0.001), and increased inotropy (maximum rate of rise of LV pressure 2001 [345] mmHg·sec-1vs 1584 [192] mmHg·sec-1; P = 0.044). Intralipid® postconditioning triggered reactive oxygen species signalling at early reperfusion and activated protection signalling (Akt, signal transducer and activator of transcription 3, and glycogen synthase kinase 3ß) in LV tissue, recapitulating all features of ILE-mediated protection reported in small rodent hearts. CONCLUSIONS: Our data show that ILE postconditioning elicits protection signalling in large mammalian hearts while mimicking clinical conditions, and is capable of enhancing protection of DCD hearts.

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.