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Establishment of an <em>Ex Vivo</em> Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Since the establishment of lung transplantation as a therapeutic strategy for advanced lung diseases, the scientific community is faced with the problem ...

University Health Network

For patients with end-stage lung disease, lung transplantation is a lifesaving therapy. Currently however, the number of patients who require a transplant exceeds the number of donor lungs available. One of the contributing factors to this is the conservative mindset of physicians who are concerned about transplanting marginal lungs due to the potential risk of primary graft dysfunction. Ex vivo lung perfusion (EVLP) technology has allowed for the expansion of donor pool of organs by enabling assessment and reconditioning of these marginal grafts before transplant. Ongoing efforts to optimize the therapeutic potential of EVLP are underway. Researchers have adopted the use of different large and small animal models to generate translational preclinical data. This includes the use of rejected human lungs, pig lungs, and rat lungs. In this review, we summarize some of the key current literature studies relevant to each of the major EVLP model platforms and identify the advantages and disadvantages of each platform. The review aims to guide investigators in choosing an appropriate species model to suit their specific goals of study, and ultimately aid in translation of therapy to meet the growing needs of the patient population.

Ex vivo lung perfusion for donor lung assessment and repair: a review of translational interspecies models.

High-dose nitric oxide (NO) has been shown effective against a variety of micro-organisms in vitro, including common bacteria found in donor organs. However, clinical obstacles related to its implementation in vivo are the formation of methemoglobin and the accumulation of toxic nitrogen compounds. Ex vivo lung perfusion (EVLP) is a platform that allows for organ maintenance with an acellular perfusion solution, thus overcoming these limitations. The present study explores the safety of continuous high-dose inhaled (iNO) during EVLP for an extended period of 12 hours.

Safety of continuous 12-hour delivery of antimicrobial doses of inhaled nitric oxide during ex vivo lung perfusion.

Cold static preservation on ice (~4°C) remains the clinical standard of donor organ preservation. However, mitochondrial injury develops during prolonged storage, which limits the extent of time that organs can maintain viability. We explored the feasibility of prolonged donor lung storage at 10°C using a large animal model and investigated mechanisms related to mitochondrial protection. Functional assessments performed during ex vivo lung perfusion demonstrated that porcine lungs stored for 36 hours at 10°C had lower airway pressures, higher lung compliances, and better oxygenation capabilities, indicative of better pulmonary physiology, as compared to lungs stored conventionally at 4°C. Mitochondrial protective metabolites including itaconate, glutamine, and -acetylglutamine were present in greater intensities in lungs stored at 10°C than at 4°C. Analysis of mitochondrial injury markers further confirmed that 10°C storage resulted in greater protection of mitochondrial health. We applied this strategy clinically to prolong preservation of human donor lungs beyond the currently accepted clinical preservation limit of about 6 to 8 hours. Five patients received donor lung transplants after a median preservation time of 10.4 hours (9.92 to 14.8 hours) for the first implanted lung and 12.1 hours (10.9 to 16.5 hours) for the second. All have survived the first 30 days after transplantation. There was no grade 3 primary graft dysfunction at 72 hours after transplantation, and median post-transplant mechanical ventilation time was 1.73 days (0.24 to 6.71 days). Preservation at 10°C could become the standard of care for prolonged pulmonary preservation, providing benefits to both patients and health care teams.

Static lung storage at 10°C maintains mitochondrial health and preserves donor organ function.

Effective treatment of respiratory infections continues to be a major challenge. In high doses (≥160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent, including its efficacy in vitro for coronavirus family. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb <6%. Both groups were ventilated continuously for 6 hours, then the animals were weaned from sedation, mechanical ventilation and followed for 3 days. During treatment, and on the third post-operative day, physiologic assessments were performed to monitor lung function and other significative markers were assessed for potential pulmonary or systemic injury. No significant change in lung function, or inflammatory markers were observed during the study period. Both gas exchange function, lung tissue cytokine analysis and histology were similar between treated and control animals. During treatment, levels of metHb were maintained <6% by administration of MB, and NO2 remained <5 ppm. Additionally, considering extrapulmonary effects, no significant changes were observed in biochemistry markers. Our findings showed that high-dose iNO delivered continuously over 6 hours with adjuvant MB is clinically feasible and safe. These findings support the development of investigations of continuous high-dose iNO treatment of respiratory tract infections, including SARS-CoV-2.

A novel pre-clinical strategy to deliver antimicrobial doses of inhaled nitric oxide.

Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP).

Ex vivo treatment of cytomegalovirus in human donor lungs using a novel chemokine-based immunotoxin.

Donor organ allocation is dependent on ABO matching, restricting the opportunity for some patients to receive a life-saving transplant. The enzymes FpGalNAc deacetylase and FpGalactosaminidase, used in combination, have been described to effectively convert group A (ABO-A) red blood cells (RBCs) to group O (ABO-O). Here, we study the safety and preclinical efficacy of using these enzymes to remove A antigen (A-Ag) from human donor lungs using ex vivo lung perfusion (EVLP). First, the ability of these enzymes to remove A-Ag in organ perfusate solutions was examined on five human ABO-A1 RBC samples and three human aortae after static incubation. The enzymes removed greater than 99 and 90% A-Ag from RBCs and aortae, respectively, at concentrations as low as 1 μg/ml. Eight ABO-A1 human lungs were then treated by EVLP. Baseline analyses of A-Ag in lungs revealed expression predominantly in the endothelial and epithelial cells. EVLP of lungs with enzyme-containing perfusate removed over 97% of endothelial A-Ag within 4 hours. No treatment-related acute lung toxicity was observed. An ABO-incompatible transplant was then simulated with an ex vivo model of antibody-mediated rejection using ABO-O plasma as the surrogate for the recipient circulation using three donor lungs. The treatment of donor lungs minimized antibody binding, complement deposition, and antibody-mediated injury as compared with control lungs. These results show that depletion of donor lung A-Ag can be achieved with EVLP treatment. This strategy has the potential to expand ABO-incompatible lung transplantation and lead to improvements in fairness of organ allocation.

Ex vivo enzymatic treatment converts blood type A donor lungs into universal blood type lungs.

Donor lungs from the United States can be offered by US organ procurement organizations to Canada if no American centers accept them. The purpose of this study is to evaluate outcomes of patients undergoing transplant at a single center in Canada using declined lungs from the United States and to compare these outcomes to patients receiving lungs from Canadian donors.

Outcomes of lung transplantation at a Canadian center using donors declined in the United States.

Since the designation of nitric oxide as "Molecule of the Year" in 1992, the scientific and clinical discoveries concerning this biomolecule have been greatly expanding. Currently, therapies enhancing the release of endogenous nitric oxide or the direct delivery of the exogenous compound are recognized as valuable pharmacological treatments in several disorders. In particular, the administration of inhaled nitric oxide is routinely used to treat patients with pulmonary hypertension or refractory hypoxemia. More recently, inhaled nitric oxide has been studied as a promising antimicrobial treatment strategy against a range of pathogens, including resistant bacterial and fungal infections of the respiratory system. Pre-clinical and clinical findings have demonstrated that, at doses greater than 160 ppm, nitric oxide has antimicrobial properties and can be used to kill a broad range of infectious microorganisms. This review focused on the mechanism of action and current evidence from in vitro studies, animal models and human clinical trials of inhaled high-dose nitric oxide as an innovative antimicrobial therapy for lung infections.

High Doses of Inhaled Nitric Oxide as an Innovative Antimicrobial Strategy for Lung Infections.

Cold static preservation (CSP) at higher temperatures (10°C) has been recently shown as an optimal strategy up to 24-36h of preservation. Here, we hypothesized that alternating 10°C static storage with cycles of normothermic ex vivo lung perfusion (EVLP) would provide conditions for cellular "recharge", allowing for multi-day lung preservation.

Successful 3-day lung preservation using a cyclic normothermic ex vivo lung perfusion strategy.

Adjuvant chemotherapy after pulmonary metastasectomy for colorectal cancer may reduce recurrence and improve survival rates; however, the benefits of this treatment are limited by the significant side effects that accompany it. The development of a novel  lung perfusion (IVLP) platform would permit the localized delivery of high doses of chemotherapeutic drugs to target residual micrometastatic disease. Nonetheless, it is critical to continuously monitor the levels of such drugs during IVLP administration, as lung injury can occur if tissue concentrations are not maintained within the therapeutic window. This paper presents a simple chemical-biopsy approach based on sampling with a small nitinol wire coated with a sorbent of biocompatible morphology and evaluates its applicability for the near-real-time  determination of oxaliplatin (OxPt) in a 72-h porcine IVLP survival model. To this end, the pigs underwent a 3-h left lung IVLP with 3 doses of the tested drug (5, 7.5, and 40 mg/L), which were administered to the perfusion circuit reservoir as a bolus after a full perfusion flow had been established. Along with OxPt levels, the biocompatible solid-phase microextraction (SPME) probes were employed to profile other low-molecular-weight compounds to provide spatial and temporal information about the toxicity of chemotherapy or lung injury. The resultant measurements revealed a rather heterogeneous distribution of OxPt (over the course of IVLP) in the two sampled sections of the lung. In most cases, the OxPt concentration in the lung tissue peaked during the second hour of IVLP, with this trend being more evident in the upper section. In turn, OxPt in supernatant samples represented ∼25% of the entire drug after the first hour of perfusion, which may be attributable to the binding of OxPt to albumin, its sequestration into erythrocytes, or its rapid nonenzymatic biotransformation. Additionally, the Bio-SPME probes also facilitated the extraction of various endogenous molecules for the purpose of screening biochemical pathways affected during IVLP (i.e., lipid and amino acid metabolism, steroidogenesis, or purine metabolism). Overall, the results of this study demonstrate that the minimally invasive SPME-based sampling approach presented in this work can serve as (pre)clinical and precise bedside medical tool.

The metabolic fate of oxaliplatin in the biological milieu investigated during  lung perfusion using a unique miniaturized sampling approach based on solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

Our recent work has challenged 4°C as an optimal lung preservation temperature by showing storage at 10°C to allow for the extension of preservation periods. Despite these findings, the impact of 10°C storage has not been evaluated in the setting of injured donor lungs.

Evaluation of 10°C as the optimal storage temperature for aspiration-injured donor lungs in a large animal transplant model.

Cytomegalovirus infection after lung transplant is associated with increased morbidity and mortality. Inflammation, infection, and longer ischemic times are important risk factors for cytomegalovirus infection. Ex vivo lung perfusion has helped to successfully increase the use of high-risk donors over the last decade. However, the impact of ex vivo lung perfusion on post-transplant cytomegalovirus infection is unknown.

Aizhou Wang

Toronto General Hospital Research Institute

University Health Network

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Aizhou Wang

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University Health Network

Establishment of an Ex Vivo Lung Perfusion Rat Model for Translational Insights in Lung Transplantation

Toronto General Hospital Research Institute