Machine Perfusion Deters Ischemia-Related Derangement of a Rodent Free Flap: Development of a Model.

INTRODUCTION: Machine perfusion can enable isolated support of composite tissues, such as free flaps. The goal of perfusion in this setting is to preserve tissues prior to transplantation or provide transient support at the wound bed. This study aimed to establish a rodent model of machine perfusion in a fasciocutaneous-free flap to serve as an affordable testbed and determine the potential of the developed support protocol to deter ischemia-related metabolic derangement. METHODS: Rat epigastric-free flaps were harvested and transferred to a closed circuit that provides circulatory and respiratory support. Whole rat blood was recirculated for 8 h, while adjusting the flow rate to maintain arterial-like perfusion pressures. Blood samples were collected during support. Extracellular tissue lactate and glucose levels were characterized with a microdialysis probe and compared with warm ischemic, cold ischemic, and anastomosed-free flap controls. RESULTS: Maintenance of physiologic arterial pressures (85-100 mmHg) resulted in average pump flow rates of 360-430 µL/min. Blood-based measurements showed maintained glucose and oxygen consumption throughout machine perfusion. Average normalized lactate to glucose ratio for the perfused flaps was 5-32-fold lower than that for the warm ischemic flap controls during hours 2-8 (P < 0.05). CONCLUSIONS: We developed a rat model of ex vivo machine perfusion of a fasciocutaneous-free flap with maintained stable flow and tissue metabolic activity for 8 h. This model can be used to assess critical elements of support in this setting as well as explore other novel therapies and technologies to improve free tissue transfer.

Expanding the Horizons of Pre-Transplant Renal Vascular Assessment Using Ex Vivo Perfusion.

Recently, immense efforts have focused on improving the preservation of (sub)optimal donor organs by means of ex vivo perfusion, which enables the opportunity for organ reconditioning and viability assessment. However, there is still no biomarker that correlates with renal viability. Therefore, it is essential to explore new techniques for pre-transplant assessment of organ quality to guarantee successful long-term transplantation outcomes. The renal vascular compartment has received little attention in machine perfusion studies. In vivo, proper renal vascular and endothelial function is essential for maintaining homeostasis and long-term graft survival. In an ex vivo setting, little is known about vascular viability and its implications for an organ's suitability for transplant. Seeing that endothelial damage is the first step in a cascade of disruptions and maintaining homeostasis is crucial for positive post-transplant outcomes, further research is key to clarifying the (patho)physiology of the renal vasculature during machine perfusion. In this review, we aim to summarize key aspects of renal vascular physiology, describe the role of the renal vasculature in pathophysiological settings, and explain how ex vivo perfusion plays a role in either unveiling or targeting such processes. Additionally, we discuss potentially new vascular assessment tools during ex vivo renal perfusion.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood.

INTRODUCTION: Expression of human complement pathway regulatory proteins (hCPRP's) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models. Here we evaluate the hypothesis that an increased human CD46 gene dose, through homozygosity or additional expression of a second hCPRP, is associated with increased protein expression and with improved protection from injury when GTKO lung xenografts are perfused with human blood. METHODS: Twenty three GTKO lungs heterozygous for human CD46 (GTKO.heteroCD46), 10 lungs homozygous for hCD46 (GTKO.homoCD46), and six GTKO.homoCD46 lungs also heterozygous for hCD55 (GTKO.homoCD46.hCD55) were perfused with human blood for up to 4 h in an ex vivo circuit. RESULTS: Relative to GTKO.heteroCD46 (152 min, range 5-240; 6/23 surviving at 4 h), survival was significantly improved for GTKO.homoCD46 (>240 min, range 45-240, p = .034; 7/10 surviving at 4 h) or GTKO.homoCD46.hCD55 lungs (>240 min, p = .001; 6/6 surviving at 4 h). Homozygosity was associated with increased capillary expression of hCD46 (p < .0001). Increased hCD46 expression was associated with significantly prolonged lung survival (p = .048),) but surprisingly not with reduction in measured complement factor C3a. Hematocrit, monocyte count, and pulmonary vascular resistance were not significantly altered in association with increased hCD46 gene dose or protein expression. CONCLUSION: Genetic engineering approaches designed to augment hCPRP activity - increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 - were associated with prolonged GTKO lung xenograft survival. Increased expression of hCD46 was associated with reduced coagulation cascade activation, but did not further reduce complement activation relative to lungs with relatively low CD46 expression. We conclude that coagulation pathway dysregulation contributes to injury in GTKO pig lung xenografts perfused with human blood, and that the survival advantage for lungs with increased hCPRP expression is likely attributable to improved endothelial thromboregulation.

Expression of human thrombomodulin by GalTKO.hCD46 pigs modulates coagulation cascade activation by endothelial cells and during ex vivo lung perfusion with human blood.

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.

48 h Normothermic Machine Perfusion With Urine Recirculation for Discarded Human Kidney Grafts.

Normothermic machine perfusion (NMP) has reshaped organ preservation in recent years. In this preclinical study, prolonged normothermic perfusions of discarded human kidney grafts were performed in order to investigate perfusion dynamics and identify potential quality and assessment indicators. Five human discarded kidney grafts were perfused normothermically (37°C) for 48 h using the Kidney Assist device with a red-blood-cell based perfusate with urine recirculation. Perfusion dynamics, perfusate and urine composition as well as injury markers were measured and analyzed. Donor age ranged from 41 to 68 years. All but one kidney were from brain dead donors. Perfusions were performed successfully for 48 h with all discarded kidneys. Median arterial flow ranged from 405 to 841 mL/min. All kidneys excreted urine until the end of perfusion (median 0.43 mL/min at the end of perfusion). While sodium levels were consistently lower in urine compared to perfusate samples, this was only seen for chloride and potassium in kidney KTX 2. Lactate, AST, LDH as well as pro-inflammatory cytokines increased over time, especially in kidneys KTX 3 and 4. Ex vivo normothermic perfusion is able to identify patterns of perfusion, biological function, and changes in inflammatory markers in heterogenous discarded kidney grafts.

Short-term outcomes after heart transplantation using donor hearts preserved with ex vivo perfusion.

The standard Conventional Cold Storage (CCS) during heart transplantation procurement is associated with time-dependent ischemic injury to the graft, which is a significant independent risk factor for post-transplant early morbidity and mortality - especially when cold ischemic time exceeds four hours. Since 2018, Rigshospitalet (Copenhagen, Denmark) has been utilising ex vivo perfusion (Organ Care System, OCS) in selected cases. The objective of this study was to compare the short-term clinical outcomes of patients transplanted with OCS compared to CCS. Methods: This retrospective single-centre study was based on consecutive patients undergoing a heart transplant between January 2018 and April 2021. Patients were selected for the OCS group when the cold ischemic time was expected to exceed four hours. The primary outcome measure was six-month event-free survival. Results: In total, 48 patients were included in the study; nine were transplanted with an OCS heart. The two groups had no significant differences in baseline characteristics. Six-month event-free survival was 77.8% [95% CI: 54.9-100%] in the OCS group and 79.5% [95% CI: 67.8-93.2%] in the CCS group (p = 0.91). While the OCS group had a median out-of-body time that was 183 min longer (p < 0.0001), the cold ischemic time was reduced by 51 min (p = 0.007). Conclusion: In a Scandinavian setting, our data confirms that utilising OCS in heart procurement allows for a longer out-of-body time and a reduced cold ischemic time without negatively affecting safety or early post-transplant outcomes.

Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis.

Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.

Pharmacological agents for defatting livers by normothermic machine perfusion.

BACKGROUND: Ex-vivo normothermic machine perfusion (NMP) preserves the liver metabolism at 37°C and has rapidly developed as a promising approach for assessing the viability and improving the performance of organs from expanded criteria donors, including fatty liver grafts. NMP is an effective method for defatting fatty livers when combined with pharmaceutical therapies. Pharmacological agents have been shown to facilitate liver defatting by NMP. OBSERVATIONS: This systematic review summarizes available pharmacological therapies for liver defatting, with a particular emphasis on defatting agents that can be employed clinically as defatting components during liver NMP as an ex vivo translational paradigm. CONCLUSION: NMP provides an opportunity for organ treatment and can be used as a defatting platform in the future with defatting agents. Nagrath's cocktail is the most commonly used defatting cocktail in NMP; however, its carcinogenic components may limit its clinical application. Thus, the combination of a defatting cocktail with a new clinically applicable component, for example, a polyphenolic natural compound, may be a novel pharmacological option.

Donation After Circulatory Death in Heart Transplantation: History, Outcomes, Clinical Challenges, and Opportunities to Expand the Donor Pool.

Heart transplantation remains the gold-standard therapy for end-stage heart failure; the expected median survival range is 12-13 years. More than 30,000 heart transplants have been performed globally in the past decade alone. With advances in medical and surgical therapies for heart failure, including durable left ventricular assist devices, an increasing number of patients are living with end-stage disease. Last year alone, more than 2500 patients were added to the heart-transplant waitlist in the United States. Despite recent efforts to expand the donor pool, including an increase in transplantation of hepatitis C-positive and extended-criteria donors, supply continues to fall short of demand. Donation after circulatory death (DCD), defined by irreversible cardiopulmonary arrest rather than donor brain death, is widely used in other solid-organ transplants, including kidney and liver, but has not been widely adopted in heart transplantation. However, resurging interest in DCD donation and the introduction of ex vivo perfusion technology has catalyzed recent clinical trials and the development of DCD heart-transplantation programs. Herein, we review the history of DCD heart transplantation, describe the currently used procurement protocols for it and examine clinical challenges and outcomes of such a procedure.

hEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood.

INTRODUCTION: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. METHODS: Livers from GaltKO.hCD46 pigs ("2-gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. RESULTS: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p = .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p = .047). Platelet sequestration tended to be delayed in the 6-gene group (p = .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p = .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p = .058). CONCLUSIONS: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Human erythrocyte fragmentation during ex-vivo pig organ perfusion.

Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex-vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet-sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in-vivo organ xenotransplant.

Donation After Circulatory Death: Expanding Heart Transplants.

Heart failure affects 6.2 million adults in the United States (US), resulting in a decrease in quality of life. Limited options exist for the treatment of end-stage heart failure. Mechanical circulatory support and transplantation are considered when no further optimization can be obtained with medical management. Heart transplant is regarded as superior to mechanical assist devices due to a lower incidence of multiorgan dysfunction. However, transplants are limited by the availability of donor organs. Heart transplants using organs from donation after circulatory death (DCD) have blossomed globally since 2014; whereas, in the US, this method has had a slower implementation. Today, the realization of the need to increase the number of donor hearts has reinvigorated the interest in heart transplantation using DCD organs. The authors review the process and discuss the unique opportunities anesthesiologists have to impact the future success of DCD heart transplantation as it continues to expand.

Ex vivo perfusion of human lymph nodes†.

In a recent study published in The Journal of Pathology, Barrow-McGee et al described a feasibility study of a real-time ex vivo perfusion model of the axillary lymph nodes (ALNs) from breast cancer patients for immune-oncological investigations. The study showed that perfused ALNs remain viable for up to 24 h, and perfusion of therapeutic antibodies confirmed the ability to reach ALN-resident cells. This work is a highly encouraging demonstration of feasibility for further research into lymphatic system function, with applications to immune function, vaccinations, and cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Successful 18-h acellular extracorporeal perfusion and replantation of porcine limbs - Histology versus nerve stimulation.

The current standard for composite tissue preservation is static cold storage (SCS) and is limited to 6 h until irreversible muscle damage occurs. Extracorporeal perfusion (ECP) is a promising technique for prolonged preservation, however, functional results have been scarcely researched. This article assessed neuromuscular function and compared results to histological alterations to predict muscle damage after ECP. Forelimbs of twelve Dutch landrace pigs were amputated and preserved by 4 h SCS at 4-6 °C (n = 6) or 18 h mid-thermic ECP with University of Wisconsin solution (n = 6). Limbs were replanted and observed for 12 h. Sham surgery was performed on contralateral forelimbs (n = 12). Histology analysis scored four subgroups representing different alterations (higher score equals more damage). Muscle contraction after median nerve stimulation was comparable between ECP, SCS, and sham limbs (P = 0.193). Histology scores were higher in ECP limbs compared to SCS limbs (4.8 vs. 1.5, P = 0.013). This was mainly based on more oedema in these limbs. In-vivo muscle contraction was well preserved after 18 h ECP compared to short SCS, although histology seemed inferior in this group. Histology, therefore, did not correlate to muscle function at 12 h after replantation. This leads to the question whether histology or neuromuscular function is the best predictor for transplant success.

High sub-zero organ preservation: A paradigm of nature-inspired strategies.

In recent years there have been several advancements in organ preservation that have yet to see widespread clinical translation. While static cold storage (SCS) at 2 °C-4 °C continues to be the state-of-the-art strategy, it contributes to the current shortage of transplantable organs due to the limited preservation times it affords combined with the limited ability of marginal grafts to tolerate SCS. The era of optimizing storage solutions to minimize SCS-induced hypothermic injury has largely plateaued in its improvements, resulting in a shift towards the use of machine perfusion systems to provide continuous metabolic support, or the use of sub-zero storage temperatures to leverage the protection brought forth by a reduction in metabolic demand. Many of the rigors that organs are subjected to at low sub-zero temperatures (-80 °C to -196 °C) commonly used for mammalian cell preservation have yet to be surmounted, and therefore the focus of this article lies on an intermediate range of storage temperatures (0 °C to -20 °C) where much success has been seen in the past two decades. Numerous mechanisms leveraged by organisms capable of withstanding prolonged periods at these temperatures through either avoiding or tolerating the formation of ice has provided a foundation for some of the more promising efforts, and thus we aim to contextualize the translation of these nature-derived strategies to mammalian organ preservation.

RNA interference therapeutics in organ transplantation: The dawn of a new era.

RNA interference (RNAi) is a natural process through which double-stranded RNA molecules can silence the gene carrying the same code as the particular RNA of interest. In 2006, the discovery of RNAi was awarded the Nobel Prize in Medicine and its success has accumulated since. Gene silencing through RNAi has been used successfully in a broad range of diseases, and, more recently, this technique has gained interest in the field of organ transplantation. Here, genes related to ischemia-reperfusion injury (IRI) or graft rejection may be silenced to improve organ quality after transplantation. Several strategies have been used to deliver siRNA, and pretransplant machine perfusion presents a unique opportunity to deliver siRNA to the target organ during ex situ preservation. In this review, the potential of RNAi in the field of organ transplantation will be discussed. A brief overview on the discovery of RNAi, its mechanism, and limitations are included. In addition, studies using RNAi to target genes related to IRI in liver, kidney, lung, and heart transplantation are discussed.

Transient hyperthermia during oxygenated rewarming of isolated rat livers.

Pretransplant machine perfusion of donor grafts has gained clinical appreciation to improve graft function and survival after transplantation. This study was aimed as pilot investigation to evaluate the additive potential of a transient ex vivo heat shock treatment of the isolated organ during machine perfusion to further protect the graft from subsequent reperfusion injury. Rat livers were retrieved after 20 min of cardiac arrest and preserved for 18 h by cold storage in HTK solution. Prior to reperfusion, livers were subjected to 2 h of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35 °C. In half of the livers (n = 7), a brief hyperthermic impulse (10 min perfusion at 42 °C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Induction of heat shock protein 70 was followed on the mRNA and protein level. Chaperone induction by transient hyperthermia was associated with a significant improvement of bile production upon reperfusion and significantly reduced enzyme loss of mitochondrial GLDH. Heat shock treatment further affected pro-inflammatory upregulation in the graft in significantly reducing gene expression as wells as protein release of TNF-alpha. It is concluded, that graft conditioning by controlled hyperthermia ex vivo may represent a feasible and useful tool to improve liver recovery after preservation.

DCD and DBD lung transplantation optimized by ex vivo perfusion: What to do when the pump fails.

BACKGROUND: Ex vivo perfusion is a safe and feasible method of assessing and using high-risk donor organs. AIM: We describe a case of successfully ex vivo treated and transplanted human lung allografts. METHODS: Donor human lungs were assessed using ex vivo, our trouble shooting protocol allowed safe recovery. RESULTS: We successfully implanted our ex vivo treated organs.

Leukoreduction in ex vivo perfusion circuits: comparison of leukocyte depletion efficiency with leukocyte filters.

BACKGROUND: Leukodepletion of whole blood-based perfusates remains a challenge in experimental models of ex vivo perfusion. This study investigated the leukoreduction efficacy of the commonly used LeukoGuard LG Arterial and BC2 Cardioplegia filters. METHODS: Eleven liters of washed porcine blood was used to evaluate the filtration efficiency of LG (n = 6) and BC2 (n = 5) filters. Filter efficacy was tested by passing 1 L of washed blood through each filter. Complete blood count was performed to detect a reduction of white blood cells, red blood cells, and hemoglobin concentration. RESULTS: The BC2 Cardioplegia filter showed a significant reduction in white blood cell count (13.16 ± 4.2 × 103 cells/µL pre-filtration, 0.62 ± 0.61 cells/µL post-filtration, p = 0.005), red blood cell count (9.18 ± 0.16 × 106 cells/µL pre-filtration, 9.02 ± 0.16 × 106 cells/µL post-filtration, p = 0.012) and hemoglobin concentration (15.89 ± 0.66 g/dL pre-filtration, 15.67 ± 0.83 g/dL post-filtration, p = 0.017). Platelet reduction in the LG filter group was statistically significant (13.23 ± 13.98 × 103 cells/µL pre-filtration, 7.15 ± 3.31 × 103 cells/µL post-filtration, p = 0.029), but no difference was seen in the BC2 group. There was no significant difference in white blood cell count in the LG filter group (10.12 ± 3.0 × 103 cells/µL pre-filtration, 10.32 ± 2.44 × 103 cells/µL post-filtration, p = 0.861). CONCLUSION: Our results suggest that the LG filter should not be used in ex vivo perfusion circuits for the purpose of leukodepletion. The BC2 filter can be used in EVP circuits with flow rates of less than 350 mL/min. Alternatively, perfusate may be leukodepleted before perfusion.

Thromboxane and histamine mediate PVR elevation during xenogeneic pig lung perfusion with human blood.

BACKGROUND: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. METHODS: GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1-Benzylimidazole (1-BIA) combined with histamine receptor blocker famotidine (n = 4) or diphenhydramine (n = 6), 1-BIA alone (n = 6) or were left untreated (n = 9). RESULTS: Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4 hours. Without treatment, initial PVR elevation within the first 30 minutes resolved partially over the following hour, and increased progressively during the final 2 hours of perfusion. In contrast, 1-BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. CONCLUSION: Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation.