Biometric Profiling to Quantify Lung Injury Through Ex Vivo Lung Perfusion Following Warm Ischemia.

Standard physiologic assessment parameters of donor lung grafts may not accurately reflect lung injury or quality. A biometric profile of ischemic injury could be identified as a means to assess the quality of the donor allograft. We sought to identify a biometric profile of lung ischemic injury assessed during ex vivo lung perfusion (EVLP). A rat model of lung donation after circulatory death (DCD) warm ischemic injury with subsequent EVLP evaluation was utilized. We did not observe a significant correlation between the classical physiological assessment parameters and the duration of the ischemic. In the perfusate, solubilized lactate dehydrogenase (LDH) as well as hyaluronic acid (HA) significantly correlated with duration of ischemic injury and length of perfusion ( p < 0.05). Similarly, in perfusates, the endothelin-1 (ET-1) and Big ET-1 correlated ischemic injury ( p < 0.05) and demonstrated a measure of endothelial cell injury. In tissue protein expression, heme oxygenase-1 (HO-1), angiopoietin 1 (Ang-1), and angiopoietin 2 (Ang-2) levels were correlated with the duration of ischemic injury ( p < 0.05). Cleaved caspase-3 levels were significantly elevated at 90 and 120 minutes ( p < 0.05) demonstrating increased apoptosis. A biometric profile of solubilized and tissue protein markers correlated with cell injury is a critical tool to aid in the evaluation of lung transplantation, as accurate evaluation of lung quality is imperative and improved quality leads to better results. http://links.lww.com/ASAIO/B49.

Electrical Impedance as a Noninvasive Metric of Quality in Allografts Undergoing Normothermic Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) increases the pool of suitable organs for transplant by facilitating assessment and repair at normothermia, thereby improving identification of quality of marginal organs. However, there exists no current objective approach for assessing total organ edema. We sought to evaluate the use of electrical impedance as a metric to assess total organ edema in lungs undergoing EVLP. Adult porcine lungs (40 kg) underwent normothermic EVLP for 4 hours. To induce varying degrees of lung injury, the allografts were perfused with either Steen, a modified cell culture media, or 0.9% normal saline. Physiologic parameters (peak airway pressure and compliance), pulmonary artery and left atrial blood gases, and extravascular lung water measurements were evaluated over time. Wet-to-dry ratios were evaluated postperfusion. Modified Murray scoring was used to calculate lung injury. Impedance values were associated with lung injury scores ( p = 0.007). Peak airway pressure ( p = 0.01) and PaO 2 /FiO 2 ratios ( p = 0.005) were both significantly associated with reduced impedance. Compliance was not associated with impedance ( p = 0.07). Wet/dry ratios were significantly associated with impedance and Murray Scoring within perfusion groups of Steen, Saline, and Modified Cell Culture ( p = 0.0186, 0.0142, 0.0002, respectively). Electrical impedance offers a noninvasive modality for measuring lung quality as assessed by tissue edema in a porcine model of normothermic EVLP. Further studies evaluating the use of impedance to assess organ edema as a quality marker in human clinical models and abdominal organs undergoing ex vivo perfusion warrant investigation.

Pegylated-Catalase Is Protective in Lung Ischemic Injury and Oxidative Stress.

BACKGROUND: Lung transplant ischemia-reperfusion injury is typified by toxic metabolites and oxygen free radicals leading to worse graft function. Catalase is an enzyme involved in oxidative-stress detoxification. We hypothesize that direct delivery of highly concentrated polyethylene glycol-catalase (PEG-CAT) during normothermic ex vivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. METHODS: To demonstrate protection, primary culture porcine endothelial cells were treated with PEG-CAT (0 to 1250 U/mL) in a model of oxidative stress (400 µM H2o2). In vivo, rat lungs were subjected to 0 hours or 1 hour of warm ischemic injury and 2 hours of EVLP with or without PEG-CAT. Perfusate was collected throughout the perfusion duration and tissue was collected at the end. Tissue and perfusate underwent analysis for markers of apoptosis and a biometric signature of lung health. RESULTS: Uptake of PEG-CAT into primary endothelial cells was demonstrated with Alexa Fluor 488-labeled PEG-CAT. Oxidatively stressed cells pretreated with PEG-CAT had significantly decreased cytotoxicity and caspase 3/7 activity and increased cell viability and cell membrane integrity. In a rat model of warm ischemia with EVLP, PEG-CAT improved allograft viability as measured by indications of cell membrane integrity (lactate dehydrogenase and hyaluronic acid), presence of vasoconstrictive peptides (endothelin-1 and big endothelin-1) released from endothelial cells, and reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling). CONCLUSIONS: In vitro and ex vivo, PEG-CAT protects against oxidative stress-induced cytotoxicity, maintains cellular metabolism, and mitigates lung ischemia-reperfusion in an experimental model. Together, these data suggest that PEG-CAT is a potential therapeutic target for donor organs at risk for ischemia-reperfusion injury.

A Novel Negative Pressure-Flow Waveform to Ventilate Lungs for Normothermic Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) is increasingly used to treat and assess lungs before transplant. Minimizing ventilator induced lung injury (VILI) during EVLP is an important clinical need, and negative pressure ventilation (NPV) may reduce VILI compared with conventional positive pressure ventilation (PPV). However, it is not clear if NPV is intrinsically lung protective or if differences in respiratory pressure-flow waveforms are responsible for reduced VILI during NPV. In this study, we quantified lung injury using novel pressure-flow waveforms during normothermic EVLP. Rat lungs were ventilated-perfused ex vivo for 2 hours using tidal volume, positive end-expiratory pressure (PEEP), and respiratory rate matched PPV or NPV protocols. Airway pressures and flow rates were measured in real time and lungs were assessed for changes in compliance, pulmonary vascular resistance, oxygenation, edema, and cytokine secretion. Negative pressure ventilation lungs demonstrated reduced proinflammatory cytokine secretion, reduced weight gain, and reduced pulmonary vascular resistance (p < 0.05). Compliance was higher in NPV lungs (p < 0.05), and there was no difference in oxygenation between the two groups. Respiratory pressure-flow waveforms during NPV and PPV were significantly different (p < 0.05), especially during the inspiratory phase, where the NPV group exhibited rapid time-dependent changes in pressure and airflow whereas the PPV group exhibited slower changes in airflow/pressures. Lungs ventilated with PPV also had a greater transpulmonary pressure (p < 0.05). Greater improvement in lung function during NPV EVLP may be caused by favorable airflow patterns and/or pressure dynamics, which may better mimic human respiratory patterns.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications - the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.