Development and prevention of ischemic contracture ("stone heart") in the pig heart.

Stone heart (ischemic contracture) is a rare and serious condition observed in the heart after periods of warm ischemia. The underlying mechanisms are largely unknown and treatment options are lacking. In view of the possibilities for cardiac donation after circulatory death (DCD), introducing risks for ischemic damage, we have investigated stone heart in pigs. Following cessation of ventilation, circulatory death (systolic pressure <8 mmHg) occurred within 13.1 ± 1.2 min; and a stone heart, manifested with asystole, increased left ventricular wall thickness and stiffness, established after a further 17 ± 6 min. Adenosine triphosphate and phosphocreatine levels decreased by about 50% in the stone heart. Electron microscopy showed deteriorated structure with contraction bands, Z-line streaming and swollen mitochondria. Synchrotron based small angle X-ray scattering of trabecular samples from stone hearts revealed attachment of myosin to actin, without volume changes in the sarcomeres. Ca2+ sensitivity, determined in permeabilized muscle, was increased in stone heart samples. An in vitro model for stone heart, using isolated trabecular muscle exposed to hypoxia/zero glucose, exhibited the main characteristics of stone heart in whole animals, with a fall in high-energy phosphates and development of muscle contracture. The stone heart condition in vitro was significantly attenuated by the myosin inhibitor MYK-461 (Mavacamten). In conclusion, the stone heart is a hypercontracted state associated with myosin binding to actin and increased Ca2+ sensitivity. The hypercontractile state, once developed, is poorly reversible. The myosin inhibitor MYK-461, which is clinically approved for other indications, could be a promising venue for prevention.

Pro-IL-1ß Is an Early Prognostic Indicator of Severe Donor Lung Injury During Ex Vivo Lung Perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to evaluate and recondition extended criteria donor lungs for transplantation. Interleukin-1ß (IL-1ß) has been identified as a prognostic indicator of nonrecovery during EVLP. This may be an effect of inflammasome activation or cellular necrosis following donation and graft preservation. Delineating the mechanism of IL-1ß release is required. METHODS: The inactive intracellular precursor molecule, pro-IL-1ß, was characterized along with the pro-IL-1ß processing enzyme, caspase-1, in the perfusate of n = 20 human lungs that had undergone EVLP (n = 10 lungs that failed to recover and were discarded versus n = 10 lungs that reconditioned and were transplanted). In an experimental porcine model, n = 8 lungs underwent EVLP and were randomized to receive either a specific NLRP3 inflammasome inhibitor or control. RESULTS: Significant increases in pro-IL-1ß and caspase-1 were observed in the perfusate from human lungs that did not recondition during EVLP compared with those that successfully reconditioned and were used for transplantation. Within the porcine EVLP, NLRP3 inflammasome inhibition reduced IL-1ß within the perfusate compared with controls, but this had no impact on lung function, hemodynamics, or inflammation. CONCLUSIONS: Our data suggest that pro-IL-1ß is passively released following cellular necrosis of the donor lung.

Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation.

BACKGROUND: Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non-ischemic perfusion for our ongoing pig-to-baboon cardiac xenotransplantation project. METHODS: Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1- KO/hCD46/hTBM) as donors and captive-bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non-ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin-containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti-non-Gal-antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions. RESULTS: In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti-non-Gal-antibodies were similar in recipients receiving grafts from either IC or CP preservation. CONCLUSIONS: While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi-organ failure in more than half of the xenotransplantation experiments. In contrast, cold non-ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long-term results after cardiac xenotransplantation.

Heparin does not improve graft function in uncontrolled non-heart-beating lung donation: an experimental study in pigs.

OBJECTIVES: Non-heart-beating donation (NHBD) has the potential to increase the number of patients treated with lung transplantation. Our study investigated, in a simulated clinical situation in the uncontrolled NHBD setting, whether or not heparin administration after death affects the donor lung function. METHODS: Twelve Swedish domestic pigs underwent ventricular fibrillation and were left untouched for 7 min followed by cardiopulmonary resuscitation with mechanical compressions for 20 min. The animals were declared dead after a 'hands-off' period of 10 min and randomized to heparin (300 IU/kg) or placebo given into a central venous catheter. In the animals receiving heparin, 2 more minutes of chest compression followed. Intrapleural cooling was initiated 1 h after death, and prevailed for 2 h. Ex vivo lung perfusion (EVLP) was performed with the Vivoline(®) system. Lung function was evaluated with blood gases at different oxygen levels, pulmonary vascular resistance (PVR), wet/dry weight ratio, macroscopic appearance and histology. RESULTS: During EVLP, there were no significant differences between groups in PaO(2) or PVR at any investigated FiO(2) level (1.0, 0.5 or 0.21). At FiO(2) 1.0 the PaO(2) in the heparin group was 64 ± 2 (range 57-73) kPa and in the non-heparin group 63 ± 4 (range 51-71) kPa. The values for PVR were 592 ± 90 (range 402-1007) and 647 ± 97 (range 426-1044), respectively. There was no significant difference between groups in wet/dry ratio or histology. CONCLUSIONS: The use of heparin is of no obvious benefit to the donor lungs in the uncontrolled NHBD situation. The exclusion of heparin will simplify lung donation from NHBDs.

Comparative outcome of double lung transplantation using conventional donor lungs and non-acceptable donor lungs reconditioned ex vivo.

A method to evaluate and recondition lungs ex vivo has been tested on donor lungs that have been rejected for transplantation. In the present paper, we compare early postoperative course between the six patients who received reconditioned lungs and the patients who received conventional donor lungs during the same period of time. During 2006 and 2007, a total of 21 patients underwent double sequential lung transplantation at the University Hospital of Lund. Six of those patients received reconditioned lungs. The other 15 patients received conventional donor lungs for transplantation without reconditioning ex vivo. The results are presented as median and interquartile range. Time in intensive care unit (days) between recipients of reconditioned lungs [13 (5-24) days], and recipients of conventional donor lungs [7 (5-12) days], P=0.44. Total hospital stay after transplantation (days) between recipients of reconditioned lungs [52 (47-60) days] and recipients of conventional donor lungs [44 (37-48) days], P=0.9. Ex vivo lung evaluation and reconditioning might not prolong early postoperative course in double lung transplantation. However, given the small number of patients, there might be a failure to detect a difference between the two groups.

Clinical transplantation of initially rejected donor lungs after reconditioning ex vivo.

BACKGROUND: A major problem in clinical lung transplantation is the shortage of donor lungs. Only about 20% of donor lungs are accepted for transplantation. A method to evaluate and recondition lungs ex vivo has been tested on donor lungs that have been rejected for transplantation. METHODS: The donor lungs were reconditioned ex vivo in an extracorporeal membrane oxygenation (ECMO) circuit with STEEN solution (Vitrolife AB, Kungsbacka, Sweden) mixed with erythrocytes. The hyperoncotic solution dehydrates edematous lung tissue. Functional evaluations were performed with deoxygenated perfusate by varying the inspired fraction of oxygen. After the reconditioning, the lungs were kept immersed at 8 degrees C in extracorporeal membrane oxygenation until transplantation was performed. RESULTS: Six of nine initially rejected donor lungs were reconditioned to acceptable function, and in six recipients, double lung transplantation was performed. Three-month survival was 100%. One patient has since died due to sepsis after 95 days, and one due to rejection after 9 months. Four recipients are alive and well without any sign of bronchiolitis obliterans syndrome 24 months after the transplantation. CONCLUSIONS: The result from the present study is promising, and we continue to transplant reconditioned lungs.

First human transplantation of a nonacceptable donor lung after reconditioning ex vivo.

PURPOSE: This article describes an ex vivo method to recondition and transplant rejected donor lungs. DESCRIPTION: A 19-year-old man was brain dead after a traffic accident. A roentgenogram showed bilateral lung contusion. He had ongoing intratracheal bleeding. After optimizing ventilator treatment and suctioning the airways, PaO2 was 9 kPa (67.5 mm Hg) on FiO2 = 0.7. The lungs were rejected by all transplantation centers in the Nordic countries. We harvested the lungs for research. The right lung was severely injured. The left lung was edematous with bleeding spots in the lower lobe, and the mediobasal segment was atelectatic. The left lung was reconditioned ex vivo and kept in topical extracorporeal membrane oxygenation until it was transplanted into a 70-year-old man with chronic obstructive pulmonary disease 17 hours later. EVALUATION: The transplanted lung functioned very well, and the patient recovered uneventfully. At 3 months control, a computed tomographic thoracic scan and transbronchial biopsies showed a normal left lung, and the patient was in very good clinical condition, only to succumb to death from unrelated events 11 months after the transplantation. CONCLUSIONS: Rejected donor lungs may be successfully transplanted after being reconditioned ex vivo.

Ex vivo evaluation of nonacceptable donor lungs.

BACKGROUND: Only a minority of the potential candidates for lung donation are considered suitable, using current evaluation methods. A new method for ex vivo evaluation, with the potential for reconditioning of marginal and nonacceptable lungs, has been developed. This is a report of the ex vivo evaluation of six donor lungs deemed nonacceptable (arterial oxygen pressure less than 40 kPa) by the Scandiatransplant, Eurotransplant, and UK transplant organizations. METHODS: The lungs are perfused ex vivo with Steen solution, a lung evaluation-preservation solution, mixed with red blood cells to a hematocrit of 15%. This extracellular solution is designed to have an optimal colloid osmotic pressure so that physiologic pressure and flow can be maintained without development of pulmonary edema. An oxygenator connected to the extracorporeal circuit maintains a normal mixed venous blood gas level in the perfusate. The lungs are ventilated and evaluated through analyses of pulmonary vascular resistance, oxygenation capacity, and arterial carbon dioxide pressure minus end-tidal carbon dioxide difference. RESULTS: The arterial oxygen pressure (inspired oxygen fraction, 1.0) increased from 27 kPa (range, 17 to 34 kPa) in situ in the organ donor at the referring hospital to 57 kPa (range, 39 to 66 kPa) during the ex vivo evaluation. The pulmonary vascular resistance varied from 3.2 to 5.7 Wood units, and the arterial carbon dioxide pressure minus end-tidal carbon dioxide difference was in the range of 1 to 2.5 kPa. CONCLUSIONS: The arterial oxygen pressure improves significantly in this model. This ex vivo evaluation model is a valuable addition to the armamentarium in finding acceptable lungs in a donor population with inferior arterial oxygen pressure values.

Transplantation of lungs from non-heart-beating donors after functional assessment ex vivo.

BACKGROUND: If lungs from patients dying of heart attacks are to serve as donor organs in a safe way, their function should be properly assessed before transplantation. The aim of this study was to investigate donor lung function evaluation in a realistic large animal model. METHODS: Twelve 60-kg pigs were used. Five minutes after ventricular fibrillation was induced, cardiopulmonary resuscitation was initiated and maintained for 20 minutes. After a 10-min hands-off period, heparin was administered through a central venous catheter followed by 20 chest compressions. Intrapleural cooling was initiated after 65 minutes of warm ischemia. Cooling proceeded for 6 hours within the cadaver, after which lung function was assessed ex vivo. Recipient pigs underwent left lung transplantation followed by right pneumonectomy, thus making these animals 100% dependent for their survival on the function of the donor lungs. RESULTS: The assessment showed that all lungs had adequate function to serve as donor lungs. All recipient animals were in good condition during the 24-hour observation period after the operation. The blood gas function did not differ significantly from that in the healthy donor animals before induction of ventricular fibrillation; pulmonary vascular resistance was within normal range. CONCLUSIONS: Lungs from non-heart-beating donors topically cooled in situ for 6 hours after 65 minutes of warm ischemia were assessed ex vivo and found to have normal function. They were then transplanted and retained normal function during a 24-hour observation period.