Hours Of Ex Vivo Lung Perfusion Research Articles

217.7: Neutrophil Extracellular Traps Removal Using Ex Vivo Lung Perfusion Restored Lung Function in Gastric Aspiration Damaged Porcine Lungs

Background: Lung transplantation (LTx) is an established therapeutic option for end-stage pulmonary disease. However, it remains hampered by donor lung scarcity. Treatments which could regenerate lungs rejected for transplant should be explored in order to combat waiting list mortality. Aspiration induced acute lung injury (ALI) is one of the contributes to the low utilization of donor lungs. Neutrophil extracellular traps (NETs) are implicated in the inflammation profile of ALI. Reduction of NETs could be used as a therapeutic option with the arrival of devices specific to their removal. NucleoCapture device selectively removes NETs from the blood by utilizing human histone H1.3 protein conjugated to polymer beads. This study investigated the impact of the NETs capture device during ex vivo lung perfusion (EVLP) on ALI damaged lungs. Methods: Healthy pigs (n=12) were stratified into two groups, treated (n=6) and not treated (n=6). All animals received 4ml/kg body weight gastric content distributed equally between the different lung segments using a bronchoscopy to induce acute lung injury. Mild to moderate ARDS was developed over 6 hours and confirmed via blood gas values, chest x-ray imaging and histological examination. Lungs were subsequently explanted en bloc and placed on EVLP for 4 hours. Treated lungs were placed in line with a NucleoCapture device connected to the EVLP circuit and the non-treated group underwent the same EVLP protocol without the device. Results: Aspiration induced ALI was induced in all subjects as confirmed by infiltration on chest x-ray, histopathological examination and by PaO2/FiO2 ratio. Following treatment with the NucleoCapture device during 4 hours of EVLP, the PaO2/FiO2 ratio was significantly increased compared to non-treated lungs and was found to surpass the threshold values suitable for transplantation. Furthermore, macroscopic evaluation of the treated lungs demonstrated improvement relative to both the initial injury as well as comparison with the non-treated cohort. Conclusions: Removal of NETs using the NucleoCapture device did restored lung function in aspiration damaged lungs. The amelioration of lung function by removing NETs shows potential clinical use of the device to increase the donor lung pool for lung transplantation.

Nicotinamide Adenine Dinucleotide (NAD+) improves lung function in rat lung ex-vivo lung perfusion model

Abstract Objective Ischemia-reperfusion injury compromises short- and long-term outcome after lung transplantation. The scarce existing data on the natural co-enzyme NAD+ suggest an antagonistic effect on hypoxia induced vasoconstriction, removal capacity on reactive oxygen species, and anti-inflammatory effects. We therefore investigated the impact of NAD+ on ischemic rat lungs during ex-vivo lung perfusion (EVLP). Methods Lungs were retrieved from 12 outbred Sprague Dawley male rats and exposed to 14 hours of cold ischemic storage. All lungs were then perfused in a rat EVLP system for 4 hours. Lung grafts were injected after 1, 2 and 3 hours with 2000 uM NAD + (N = 6) or placebo (N = 6) in the perfusate in proximity of the pulmonary artery. EVLP physiology and biochemistry were monitored. Results During the 4 hours of EVLP, the lung function increased significantly in the NAD+ group when compared to the placebo group. We monitored a higher vascular flow (p = 0.018), a lower mean pulmonary pressure (p = 0.007) and increased oxygenation capacity (p = 0.003). Lung compliance and weight were comparable. Tissue inflammation measured by myeloperoxidase was significantly lower in the NAD+ group (p = 0.015). In the perfusate, we observed in the NAD+ group significantly lower levels of pro-inflammatory interleukin-18 (p = 0.033) and a trend towards high levels of anti-inflammatory interleukin-10 (p = 0.080) and low levels of pro-inflammatory interleukin-12 (p = 0.146). Conclusion Findings from this preliminary study demonstrated that NAD+ is a promising agent with both anti-inflammatory properties and the ability to improve ischemic lung function. This observation should be validated in a large animal model.

Nicotinamide Adenine Dinucleotide (NAD+) Improves Lung Function in Rat Lung Ex-Vivo Lung Perfusion Model

Purpose Ischemia-reperfusion injury compromises short- and long-term outcome after lung transplantation. The scarce existing data on the natural co-enzyme NAD+ suggest an antagonistic effect on hypoxia induced vasoconstriction, removal capacity on reactive oxygen species, and anti-inflammatory effects. We therefore investigated the impact of NAD+ on ischemic rat lungs during ex-vivo lung perfusion (EVLP). Methods Lungs were retrieved from 12 outbred Sprague Dawley male rats and exposed to 14 hours of cold ischemic storage. All lungs were then perfused in a rat EVLP system for 4 hours. Lung grafts were injected after 1, 2 and 3 hours with 2000 uM NAD+ (N=6) or placebo (N=6) in the perfusate in proximity of the pulmonary artery. EVLP physiology and biochemistry were monitored. Results During the 4 hours of EVLP, the lung function increased significantly in the NAD+ group when compared to the placebo group. We monitored a higher vascular flow (p=0.018), a lower mean pulmonary pressure (p=0.007) and increased oxygenation capacity (p=0.003). Lung compliance and weight were comparable. Tissue inflammation measured by myeloperoxidase was significantly lower in the NAD+ group (p=0.015). In the perfusate, we observed in the NAD+ group significantly lower levels of pro-inflammatory interleukin-18 (p=0.033) and a trend towards high levels of anti-inflammatory interleukin-10 (p=0.080) and low levels of pro-inflammatory interleukin-12 (p=0.146). Conclusion Findings from this preliminary study demonstrated that NAD+ is a promising agent with both anti-inflammatory properties and the ability to improve ischemic lung function. This observation should be validated in a large animal model.

Evidence of Air Trapping During Ex Vivo Lung Perfusion: A Swine Experimental Lung Imaging and Mechanics Study

Ex vivo lung perfusion (EVLP) allows the ventilation and perfusion of lungs to evaluate their viability for transplantation. The aim of this study is to compare the mechanical, morphologic and functional properties of lungs during EVLP with values obtained in vivo to guide a safe mechanical ventilation strategy.Lungs from 5 healthy pigs were studied in vivo and during 4 hours of EVLP. Lung compliance, airway resistance, gas exchange, and hemodynamic parameters were collected at positive end-expiratory pressure (PEEP) of 5 cm H2O. Computed tomography was performed at PEEP 0, PEEP 5, and total lung capacity (TLC). Lung pressure-volume (PV) curves were performed from PEEP 0 to TLC.Lung compliance decreased during EVLP (53 ± 5 mL/cm H2O vs 29 ± 7 mL/cm H2O, P < .05), and the PV curve showed a lower inflection point. Gas content (528 ± 118 mL vs 892 ± 402 mL at PEEP 0) and airway resistance (25 ± 5 vs 44 ± 9 cmH2O/L∗s-1, P < .05) were higher during EVLP. Alveolar dead space (5% ± 2% vs 17% ± 6%, P < .05) and intrapulmonary shunt (9% ± 2% vs 28% ± 13%, P < .05) increased ex vivo compared to in vivo, while the partial pressure of oxygen to inspired oxygen fraction ratio (PO2/FiO2) did not differ (468 ± 52 mm Hg vs 536 ± 14 mm Hg).In conclusion, during EVLP lungs show signs of air trapping and bronchoconstriction, resulting in low compliance and increased alveolar dead space. Intrapulmonary shunt is high despite oxygenation levels acceptable for transplantation.

Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1hiTissue-Resident Memory T Cells

Rationale: Tissue-resident memory T cells (TRM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (MLR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between MLR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: TRM and MLR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3+ and CD68+ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung TRM and MLR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8+ TRM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that MLR provide costimulatory signaling to PD1hi CD4+ and CD8+ lung TRM,, augmenting the effector cytokine production and degranulation of TRM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human MLR colocalize with and provide costimulation signaling to TRM, augmenting their effector function.

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

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 exvivo lung perfusion (EVLP) significantly reduces ischemia-reperfusion injury. 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). Invivo, 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. 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). Invitro and exvivo, 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.

Ex Vivo Lung Perfusion Improves the Inflammatory Signaling Profile of the Porcine Donor Lung Following Transplantation.

Primary graft dysfunction and allograft rejection represent major caveats to successful lung transplantation. Reducing inflammation in donor lungs before transplantation may improve outcomes. Evidence exists that ex vivo lung perfusion (EVLP) can alter the donor lung environment, although the mechanisms remain unclear. This study aimed to characterize the inflammatory signaling profile of the lung following standard and EVLP transplant and delineate the immediate impact on the recipient circulation. Female recipient pigs (n = 12) were randomized to undergo left lung transplantation from male donors either using the gold standard protocol (static cold storage) or following 3 hours of EVLP. The relative phosphorylation of 44 phosphokinases and the relative expression of 35 apoptosis-related molecules were profiled within the donor lung 24 hours posttransplantation. A global profile of mitochondrial salvage and cell survival was observed in the EVLP lung tissue compared with lungs undergoing standard transplantation. This included increased phosphorylation of downstream prosignaling kinases, including ERK1/2 and FAK. In addition, there was upregulated expression of the antiapoptotic proteins Bcl-2, HSP-70, LIVIN, and PON2 with downregulation of apoptosis inducing mitochondrial associated molecules, including clusterin, cytochrome C, and HTRA2/OMI. In the early postoperative period, there were significantly lower levels of circulating mitochondrial DNA in recipients receiving EVLP lungs compared with a standard transplant (P = 0.016). Genomic DNA did not differ between groups, with donor DNA undetectable at all time points. EVLP alters the inflammatory signaling profile of the donor lung before transplantation, with a global cell survival and antiapoptotic signature.

Mesenchymal stromal cell therapy during ex vivo lung perfusion ameliorates ischemia-reperfusion injury in lung transplantation.

The application of mesenchymal stromal cell (MSC)-based therapy during ex vivo lung perfusion (EVLP) could repair injured donor lungs before transplantation. The aim of this study was to determine the efficacy of MSC therapy performed during EVLP on ischemia-reperfusion injury using a pig lung transplant model. Following 24 hours of cold storage, pig lungs were randomly assigned to 2 groups (n = 6 each), the control group without MSC vs the MSC group, where 5 × 106 cells/kg MSCs were delivered through the pulmonary artery during EVLP. After 12 hours of EVLP, followed by a 1-hour second cold preservation period, the left lung was transplanted and reperfused for 4 hours. EVLP perfusate hepatocyte growth factor (HGF) level at 12 hours was significantly elevated in the MSC group compared with the control and was associated with a significant decrease in cell death markers, cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells, in the MSC group. The MSC group showed significantly lower interleukin (IL)-18 and interferon gamma levels and a significantly higher IL-4 level in lung tissue at 12 hours of EVLP than the control group. After transplantation, the MSC group showed a significant increase in lung tissue HGF level compared with the control group, associated with a significantly reduced lung tissue wet-to-dry weight ratio. Lung tissue tumor necrosis factor-α level and pathological acute lung injury score were significantly lower in the MSC group than the control group. The administration of MSCs ameliorated ischemic injury in donor lungs during EVLP and attenuated the subsequent ischemia-reperfusion injury after transplantation.

Better Oxygenation in Upper Lobes Than in Whole Lungs in Marginal Human Donor Lungs Utilized for Cellular Ex Vivo Lung Perfusion

Purpose Anatomical regions of marginal donor lungs display significant heterogeneity in function raising the possibility that good functioning regions might be suitable for transplantation. The aim of this study was to compare gas exchange of upper lobes, which are possibly less susceptible to injury, with whole lungs using ex vivo lung perfusion (EVLP). Methods Nine rejected donor lungs were perfused for 2 hours of cellular EVLP utilizing the Swedish protocol. Transplant suitability was evaluated based on blood gas analysis, hemodynamics, ventilator parameters, palpation, visual findings, and lung weight. In order to assess upper lobe gas exchange, blood samples from upper lobe pulmonary vein (ULPV) were taken selectively at 2 hours of EVLP. PaO2/FiO2 (P/F) ratio, shunt fraction (SF), and A-a gradient in ULPV samples were compared with those of left atrium (LA). Results Upper lobes demonstrated a significantly higher P/F ratio [median (minimum - maximum)] [396 (315-583) vs. 284 (104-415), mmHg, p = 0.02, Figure A] and lower SF [21.8 (7.3-34.6) vs. 29.1 (22.7-73.9), %, p = 0.04] than whole lungs. Based on evaluation at 2 hours of EVLP, 6 of 9 lungs were deemed to be non-suitable for transplantation. In non-suitable group, P/F ratio in ULPV was significantly higher than that in LA [439 (315-583) vs. 245 (104-415) mmHg, p = 0.02, Figure B], whereas SF and A-a gradient of ULPV were significantly lower than those of LA [SF, 18.5 (7.3-34.6) vs. 34.8 (22.7-73.9) %, p = 0.04; A-a gradient, 224.9 (83.5-347.9) vs. 418.4 (246.2-569.6) mmHg, p = 0.02]. In contrast, no difference was found between the ULPV and LA parameters in lungs deemed suitable for transplant. Conclusion These results demonstrate that in marginal donor lungs judged to be non-suitable for transplant at 2 hours of EVLP, the majority of lungs have well-functioning upper lobes. These findings suggest that, in this setting, upper lobes may be suitable for lobar transplantation which could help reduce wait-list mortality.

The Protective Effect of Prone Positioning on Human Rejected Donor Lungs during Cellular Ex Vivo Lung Perfusion

Purpose We previously reported that prone positioning (PP) of porcine lungs during cellular ex vivo lung perfusion (EVLP) diminished inflammatory injury and improved oxygenation and other pulmonary parameters during cellular EVLP. We hypothesized that prone positioning of human lungs during cellular EVLP may diminish injury and improve function, as well. The aim of this study is to validate the previous results with porcine lungs and evaluate the potential benefits of PP EVLP with human rejected donor lungs. Methods Eleven human rejected donor lungs were flushed and stored for 15-16 hours with Perfadex. Lungs were then subjected to 2 hours of cellular EVLP with either maintenance of prone positioning (prone group, n = 6) or supine positioning (control group, n = 5). Lung function was evaluated using blood gas analysis, airway and vascular parameters, visual findings, wet/dry (W/D) ratio and lung weight at 2 hours of EVLP. Results Lung weight (LW) ratio (LW at 2 hours of EVLP/ LW prior to EVLP) was lower in Prone group than in Control group [median (interquartile range)] [1.09 (0.82-1.32) vs. 1.22 (1.01-1.50), p = 0.5] (not statistical significant). The PaO2/FiO2 (P/F) ratio of left atrial blood was significantly higher in Prone group than in Control [335.5 (297.2-392.5) vs. 198.0 (190.5-271.0) mmHg, p Conclusion These results demonstrate that PP during 2 hours of cellular EVLP result in less lung weight gain, and better P/F ratio, especially in the lower lobes. These results were consistent with our previous animal study, and suggest that prone positioning EVLP of rejected human donor lungs also may diminish IRI during EVLP.

Evaluation of the Effects of the Correction of Hydroelectrolytic Disorders during Prolonged Ex-Vivo Lung Perfusion

Purpose During ex-vivo lung perfusion (EVLP), the ionic concentrations and pH of perfusion liquid are modified. In the course of this study, we evaluated whether these ionic disruptions have an impact on the lung graft during 12 hours of EVLP and whether the correction of these disruptions can avoid any lung lesions. If this were confirmed, it would allow longer EVLP time. Methods Twelve pigs' lungs were conventionally retrieved, cold preserved, perfused normothermically ex vivo for 12 hours, cold preserved for a second time and transplanted and reperfused for 4 hours. Ionic analysis and arterial and venous gas analysis were performed every hour during EVLP. Every 2 hours, 100mL of Steen solution were replaced with new Steen solution in the Steen N group and with modified Steen solution in the Steen + group. After 12 hours of EVLP, the right lung was harvested for structural, biological, cellular and histological analysis. The left lung was transplanted to a healthy swine and blood ionic and gas analysis were performed every hour during the left lung reperfusion. Results During EVLP, ∆PO2, compliance and pulmonary vascular resistance were similar in both groups. The ionic concentrations and pH were corrected in the Steen + group. After the left lung transplant, physiological and biological parameters were similar in both groups. Structural analysis by scanning electron microscopy showed a rise of interalveolar pores in the Steen + group. Conclusion The correction of hydroelectrolytic disorders during 12 hours of EVLP is feasible but does not improve lung function during EVLP and after the left lung transplant.

Reperfusion Inflammatory State of Human Lungs during Cellular Ex-Vivo Perfusion

Purpose Normothermic cellular ex-vivo lung perfusion (EVLP) can provide a platform for studying ischemia-reperfusion injury (IRI), with the donor lungs being the only source of cytokine appearance in the perfusate. The inflammatory effects of cellular EVLP on lungs are inadequately explored. Herein, we aimed to examine the reperfusion inflammatory state during cellular EVLP in relation to donor factors and impact on lung function. Methods For research purposes, 42 clinically rejected donor lungs underwent normothermic cellular EVLP with open left atrium and flow equivalent to 100% of cardiac output for 2 hours. Reasons for rejection were donor old age, smoking history, low PO2/FiO2 (P/F) ratio, or unavailability of a matched recipient. Perfusate samples were collected at the beginning of EVLP (0 hours), then at 1 and 2 hours of EVLP. Lung weight was measured at the end of EVLP as an indicator for the degree of edema. Data were presented as median (interquartile range). Results Interleukin (IL)-6, IL-8, IL-10, IL-1β, and tumor necrosis factor (TNF)-α were significantly up-regulated in the EVLP perfusate at 1 and 2 hours compared to baseline 0 hours (p Conclusion Cytokine array has shown that cellular EVLP is associated with reperfusion inflammatory state in human lungs. IL-1β and IL-10 were mostly affected by donor factors, while IL-6, IL-8, and TNF-α had the most effect on lung function during EVLP. These results suggest that cellular EVLP can be utilized to study human lung IRI and develop therapeutic targets.

Triptolide Attenuates Graft Inflammation During Ex Vivo Lung Perfusion

Purpose Ex vivo lung perfusion (EVLP) has been proposed as a platform for lung graft assessment and preservation. However, its proinflammatory milieu may be associated with deterioration in graft quality which may contribute to posttransplant graft dysfunction. Triptolide (TL), a diterpenoid triepoxide, has recently been shown to have a therapeutic potential in various disease states due to its anti-inflammatory properties. Based on this, we sought to assess the impact of TL on graft preservation during EVLP as well as associated posttransplant outcomes. Methods Using rat heart-lung blocs, EVLP was performed with Steen solution containing 100 nM of TL at 37°C for 4 hours. Graft evaluation included physiologic, metabolic and functional parameters. Tissue inflammatory profile and endoplasmic reticulum (ER) stress markers were assessed. Following 4 hours of EVLP, orthotopic single lung transplantation was performed using 3-cuff technique. Results were compared with those of lung grafts undergoing EVLP without TL. Results Physiologic and functional parameters of lung grafts on EVLP with TL were better than those without treatment (Figure A). Glucose consumption was significantly decreased in lungs treated with TL. Levels of proinflammatory cytokine mRNA were lower in TL-treated lungs which is attributed to inhibition of nuclear factor κB (NF-κB) signaling. In addition, TL improved ER stress during EVLP as evidenced by diminished ER stress markers. Most importantly, posttransplant graft function was significantly better in the TL group compared to untreated group (Figure C). Conclusion Treatment of lung grafts with TL during EVLP may serve as a potential compound for enhanced graft preservation. These studies warrant further investigation into its potential protective effect and may have implications for improving posttransplant outcomes.

Ex vivo perfusion induces a time- and perfusate-dependent molecular repair response in explanted porcine lungs.

Ex vivo lung perfusion (EVLP) shows promise in ameliorating pretransplant acute lung injury (ALI) and expanding the donor organ pool, but the mechanisms of ex vivo repair remain poorly understood. We aimed to assess the utility of gene expression for characterizing ALI during EVLP. One hundred sixty-nine porcine lung samples were collected in vivo (n = 25), after 0 (n = 11) and 12 (n = 11) hours of cold static preservation (CSP), and after 0 (n = 57), 6 (n = 8), and 12 (n = 57) hours of EVLP, utilizing various ventilation and perfusate strategies. The expression of 53 previously described ALI-related genes was measured and correlated with function and histology. Twenty-eight genes were significantly upregulated and 6 genes downregulated after 12 hours of EVLP. Aggregate gene sets demonstrated differential expression with EVLP (P < .001) but not CSP. Upregulated 28-gene set expression peaked after 6 hours of EVLP, whereas downregulated 6-gene set expression continued to decline after 12 hours. Cellular perfusates demonstrated a greater reduction in downregulated 6-gene set expression vs acellular perfusate (P < .038). Gene set expression correlated with relevant functional and histologic parameters, including P/F ratio (P < .001) and interstitial inflammation (P < .005). Further studies with posttransplant results are warranted to evaluate the clinical significance of this novel molecular approach for assessing organ quality during EVLP.

Prediction of transplant outcome after 24-hour ex vivo lung perfusion using the Organ Care System in a porcine lung transplantation model.

Ex vivo lung perfusion (EVLP) has become routine practice in lung transplantation. Still, running periods exceeding 12hours have not been undertaken clinically to date, and it remains unclear how the perfusion solution for extended running periods should be composed and which parameters may predict outcomes. Twenty-four porcine lungs underwent EVLP for 24hours using the Organ Care System (OCS). Lungs were ventilated and perfused with STEEN's solution enriched with erythrocytes (n=8), acellular STEEN's solution (n=8), or low-potassium dextran (LPD) solution enriched with erythrocytes (n=8). After 24hours, the left lungs were transplanted into recipient pigs. After clamping of the contralateral lung, the recipients were observed for 6hours. The most favorable outcome was observed in organs utilizing STEEN solution enriched with erythrocytes as perfusate, whereas the least favorable outcome was seen with LPD solution enriched with erythrocytes for perfusion. Animals surviving the observation period showed lower peak airway pressure (PAWP) and pulmonary vascular resistance (PVR) during OCS preservation. The results suggest that transplantation of lungs following 24hours of EVLP is feasible but dependent on the composition of the perfusate. PAWP and PVR during EVLP are early and late predictors of transplant outcome, respectively.

Towards donor lung recovery-gene expression changes during ex vivo lung perfusion of human lungs.

We and others have demonstrated that acellular normothermic ex vivo lung perfusion of high-risk donor lungs can result in posttransplant outcomes equivalent to that of contemporaneous lung transplantation using standard donor lungs. However, the mechanism of this effect remains unclear. Given the restoration of cellular metabolic activity during normothermic perfusion, one possibility is that of lung healing via natural innate recovery mechanisms. We explored this by examining the gene expression changes occurring in human lungs during ex vivo lung perfusion. Human lungs clinically rejected for transplantation were perfused for 12hours of EVLP with biopsies taken at the start, at 1hour, at 3hours, and then every 3hours thereafter to 12hours. Temporal changes were identified in 2585 genes using the Short Time-series Expression Miner and used for pathway analysis. Despite increases in endothelial markers of inflammation, circulating leukocyte cell-specific gene expression fell over 12hours of ex vivo lung perfusion (EVLP), suggesting an interrupted inflammation response secondary to washout of circulating leukocytes. Analysis of these gene changes suggests lung recovery follows specific stages: cellular death, cellular preservation, cellular reorganization, and cellular invasion. EVLP may improve posttransplant lung function by washout of leukocytes and facilitating innate mechanisms of repair.

A Leukocyte Filter Does Not Provide Further Benefit During Ex Vivo Lung Perfusion.

Normothermic ex vivo lung perfusion (EVLP) allows for assessment and reconditioning of donor lungs. Although a leukocyte filter (LF) is routinely incorporated into the EVLP circuit; its efficacy remains to be determined. Twelve pig lungs were perfused and ventilated ex vivo in a normothermic state for 12 hours. Lungs (n = 3) were allocated to four groups according to perfusate composition and the presence or absence of a LF in the circuit (acellular ± LF, cellular ± LF). Acceptable physiologic lung parameters were achieved during EVLP; however, increased amounts of pro-inflammatory cytokines (TNF-α and IL-6) and leukocytes in the perfusate were observed despite the presence or absence of a LF. Analysis of cells washed off the LF demonstrates that it trapped leukocytes although being ineffective throughout perfusion as it became saturated over 12 hours of EVLP. We conclude that there is no objective evidence to support the routine incorporation of a LF during EVLP as it does not provide further benefit and its removal does not appear to cause harm. The lack of hypothesized benefit to a LF may be because of the saturation of the LF with donor leukocytes, leading to similar amounts of circulating leukocytes still present in the perfusate with and without a LF.

Higher M30 and high mobility group box 1 protein levels in ex vivo lung perfusate are associated with primary graft dysfunction after human lung transplantation

Ex vivo lung perfusion (EVLP) enables assessment of marginal donor lungs for transplantation, with similar clinical outcomes to conventional lung transplantation. We investigated whether cell death-related proteins in the EVLP perfusate could predict primary graft dysfunction (PGD) after transplantation. M30 (indicating epithelial apoptosis), M65 (indicating total epithelial cell death), and high mobility group box 1 (HMGB-1, related to cell death and inflammation) protein levels in EVLP perfusate were measured by enzyme-linked immunosorbent assay and correlated with clinical outcomes. From 100 sequential EVLP patients, 79 lungs were transplanted. Patients who were bridged with extracorporeal life support (ECLS, n = 6) or who received lobar/single lung (n = 25) were excluded. PGD grade 3 (partial pressure of arterial oxygen/fraction of inspired oxygen <200 or need for ECLS) developed in 11 at any time within 72 hours after transplantation (PGD Group). PGD grade 3 did not develop in 34 patients (Control Group). M30 was significantly higher in the PGD Group than in the Control Group at 1 hour (PGD: 73.3 ± 24.9, control: 53.9 ± 15.9 U/liter; p < 0.01) and at 4 hours (PGD: 137.0 ± 146.6, Control: 72.4 ± 40.0 U/liter; p = 0.046) of EVLP. The increase of HMGB-1 from 1 to 4 hours of EVLP was significantly greater in the PGD Group (PGD: 37.0 ± 25.4, Control: 7.2 ± 16.8 ng/ml; p < 0.001). Higher levels of or a greater increase in M30 and a greater increase in HMGB-1 were associated with higher mortality in Cox regression. Levels of M30 and HMGB-1 in the EVLP perfusate correlate with PGD after lung transplantation and might therefore be useful biomarkers to improve donor lung assessment during EVLP.

Cytokine filtration modulates pulmonary metabolism and edema formation during ex vivo lung perfusion

Ex vivo lung perfusion (EVLP) has improved the process of donor lung management. Cytokine accumulation during EVLP has been shown to correlate with worse outcome after lung transplantation. Our objective in this study was to test the safety and efficacy of cytokine filtration during EVLP in a large animal model. Pig donor lungs were preserved for 24 hours at 4°C, followed by 12 hours of EVLP, according to the Toronto protocol. The perfusate was continuously run through an absorbent device (CytoSorb) via a veno-venous shunt from the reservoir in the filter group. EVLP was performed according to the standard protocol in the control group (n = 5 each). EVLP physiology, lung X-ray, perfusate biochemistry, inflammatory response and microscopic injury were assessed. Cytokine filtration significantly improved airway pressure and dynamic compliance during the 12-hour perfusion period. Lung X-rays acquired at the end of perfusion showed increased consolidation in the control group. Electrolyte imbalance, determined by increased hydrogen, potassium and calcium ion concentrations in the perfusate, was markedly worsened in the control group. Glucose consumption and lactate production were markedly reduced, along with the lactate/pyruvate ratio in the filter group. Cytokine expression profile, tissue myeloperoxidase activity and microscopic lung injury were significantly reduced in the filter group. Continuous perfusate filtration through sorbent beads is effective and safe during prolonged EVLP. Cytokine removal decreased the development of pulmonary edema and electrolyte imbalance through the suppression of anaerobic glycolysis and neutrophil activation in this setting. Further studies are needed to test the beneficial effect of cytokine filtration on post-transplant lung function.

Optimal ex vivo lung perfusion techniques with oxygenated perfusate

Accumulating evidence supports an increasing role of ex vivo lung perfusion (EVLP) in clinical lung transplantation. However, EVLP has adverse effects on the quality of lung grafts, which have rarely been discussed. Careful optimization of current EVLP protocols might improve outcomes. This study examined effects of different levels of oxygenation of the perfusate circulated through the lungs during EVLP and the impact on post-transplant functional outcomes. We compared results of 4 different oxygenation levels in the perfusate during EVLP: 6% oxygen (O2), 40% O2, 60% O2, and 100% O2. We evaluated lung function, compliance, and vascular resistance and levels of glucose and other markers in the perfusate. After EVLP, lung grafts were transplanted, and post-transplant outcomes were compared. Lungs perfused with 40% O2 on EVLP had the lowest glucose consumption compared with the other perfusates. Lungs treated with 40% O2 or 60% O2 exhibited significantly less inflammation, as indicated by reduced pro-inflammatory cytokine messenger RNA levels compared with lungs perfused with 6% O2 or 100% O2. Significantly more oxidative damage was noted after 4 hours of EVLP perfused with 100% O2. After transplantation, lungs perfused with 40% O2 during EVLP had the best post-transplant functional outcomes. Optimization of O2 levels in the perfusate during EVLP improved outcomes in this rat model. Deoxygenated perfusate, the current standard during EVLP, exhibited significantly more inflammation with compromised cellular metabolic activity and compromised post-transplant outcomes.