Hemodynamic evaluation of anesthetized baboons and piglets by transpulmonary thermodilution: Normal values and interspecies differences with respect to xenotransplantation

Background Transpulmonary thermodilution is well established as a tool for in-depth hemodynamic monitoring of critically ill patients during surgical procedures and intensive care. It permits easy assessment of graft function following cardiac transplantation and guides post-operative volume and catecholamine therapy. Since no pulmonary catheter is needed, transpulmonary thermodilution could be useful in experimental cardiac pig-to-baboon xenotransplantation. However, normal values for healthy animals have not yet been reported. Here, we present data from piglets and baboons before xenotransplantation experiments and highlight differences between the two species and human reference values. Methods Transpulmonary thermodilution from baboons (body weight 10-34 kg) and piglets (body weight 10-38kg) were analyzed. Measurements were taken in steady state after induction of general anesthesia before surgical procedures commenced. Cardiac index (CI), mean arterial pressure (MAP), systemic vascular resistance index (SVRI), parameters quantifying cardiac filling (global end-diastolic volume index, GEDI), and pulmonary edema (extravascular lung water, ELWI) were assessed. Results Preload, afterload, and contractility parameters clearly correlated with total body weight or body surface area. Baboons had lower CI values than weight-matched piglets (4.2 +/- 0.9l/min/m(2) vs 5.3 +/- 1.0/min/m(2), P < .01). MAP and SVRI were higher in baboons than piglets (MAP: 99 +/- 22 mm Hg vs 62 +/- 11 mm Hg, P < .01;SVRI: 1823 +/- 581 dyn*s/cm(5)*m(2) vs 827 +/- 204 dyn*s/cm(5)*m(2), P < .01). GEDI and ELWI did differ significantly between both species, but measurements were within similar ranges (GEDI: 523 +/- 103 mL/m(2) vs 433 +/- 78 mL/m(2), P < .01;ELWI: 10 +/- 3 mL/kg vs 11 +/- 2 mL/kg, P < .01). Regarding adult human reference values, CI was similar to both baboons and piglets, but all other parameters were different. Conclusions Parameters of preload, afterload, and contractility differ between baboons and piglets. In particular, baboons have a much higher afterload than piglets, which might be instrumental in causing perioperative xenograft dysfunction and post-operative myocardial hypertrophy after orthotopic pig-to-baboon cardiac xenotransplantation. Most transpulmonary thermodilution-derived parameters obtained from healthy piglets and baboons lie outside the reference ranges for humans, so human normal values should not be used to guide treatment in those animals. Our data provide reference values as a basis for developing algorithms for perioperative hemodynamic management in pig-to-baboon cardiac xenotransplantation

Evidence for Microchimerism in Baboon Recipients of Pig Hearts

Xenotransplantation, like allotransplantation, is usually associated with microchimerism, i.e., the presence of cells from the donor in the recipient. Microchimerism was reported in first xenotransplantation trials in humans, as well as in most preclinical trials in nonhuman primates (for review, see Denner, Viruses 2023, 15, 190). When using pigs as xenotransplantation donors, their cells contain porcine endogenous retroviruses (PERVs) in their genome. This makes it difficult to discriminate between microchimerism and PERV infection of the recipient. Here, we demonstrate the appropriate virological methods to be used for the identification of microchimerism, first by screening for porcine cellular genes, and then how to detect infection of the host. Using porcine short interspersed nuclear sequences (SINEs), which have hundreds of thousands of copies in the pig genome, significantly increased the sensitivity of the screening for pig cells. Second, absence of PERV RNA demonstrated an absence of viral genomic RNA or expression as mRNA. Lastly, absence of antibodies against PERV proteins conclusively demonstrated an absence of a PERV infection. When applying these methods for analyzing baboons after pig heart transplantation, microchimerism could be demonstrated and infection excluded in all animals. These methods can be used in future clinical trials

Glycocalyx dynamics and the inflammatory response of genetically modified porcine endothelial cells.

Xenotransplantation is a promising approach to reduce organ shortage, while genetic modification of donor pigs has significantly decreased the immunogenic burden of xenotransplants, organ rejection is still a hurdle. Genetically modified pig organs are used in xenotransplantation research, and the first clinical pig-to-human heart transplantation was performed in 2022. However, the impact of genetic modification has not been investigated on a cellular level yet. Endothelial cells (EC) and their sugar-rich surface known as the glycocalyx are the first barrier encountering the recipient's immune system, making them a target for rejection. We have previously shown that wild type venous but not arterial EC were protected against heparan sulfate (HS) shedding after activation with human serum or human tumor necrosis factor alpha (TN

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNF alpha were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs

Hemodynamics in pig‐to‐baboon heterotopic thoracic cardiac xenotransplantation: Recovery from perioperative cardiac xenograft dysfunction and impairment by cardiac overgrowth

Introduction Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. Methods We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. Results Aortic pressures and heart rates of the recipients’ hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. Conclusion The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size

Overcoming perioperative inflammation as a hurdle for successful preclinical orthotopic cardiac xenogeneic transplantations - particular in regard of the mandatory use of heart-lung machines

Introduction After orthotopic cardiac xenotransplantation, the combination of both the inflammatory responses to the exposure of a recipient to the xenogeneic organ and the use of cardiopulmonary bypass has been assumed to cause detrimental side effects. These have been described not only to affect the transplanted organ (heart) itself, but also the recipient's lungs. In this article, we summarize how these possible detrimental processes can be minimized or even avoided. Methods Data from eight pig-to-baboon orthotopic cardiac xenotransplantation experiments were analyzed with a special focus on early (within the first week) postoperative organ dysfunction and systemic inflammatory responses. Non-ischemic heart preservation and the careful management of the heart-lung machine were deemed essential to guarantee not only the immediate function of the transplanted xenogeneic organ but also the prompt recovery of the recipient. Results After weaning from cardiopulmonary bypass, very low catecholamine amounts were needed to ensure an adequate pump function and cardiac output. Central venous oxygen saturation and serum lactate levels remained within normal ranges. All animals were successfully weaned from ventilation within the first postoperative hours. Serum parameters of the transplants and native kidneys and livers were initially slightly elevated or always normal, as were hemoglobin, LDH, and platelet measurements. Markers of systemic inflammation, C-reactive protein, and IL-6 were slightly elevated, but the reactions caused no lasting damage. Conclusion Consistent short-term and long-term results were achieved after orthotopic cardiac pig-to-baboon transplantation without detrimental inflammatory responses or signs of multiorgan failure. In comparison to allogeneic procedures, non-ischemic heart preservation was important for successful immediate organ function, as was the management of the heart-lung machine. Thus, we believe that genetically modified porcine hearts are ready for use in the clinical setting

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