End Of Ex Vivo Lung Perfusion Research Articles

Lung Function and Inflammatory Profiling of Damaged Rat Donor Lungs Following EVLP Heat Stress with Different Temperatures

<h3>Purpose</h3> Ex-vivo lung perfusion (EVLP) is a validated technique allowing evaluation and reconditioning of damaged donor lungs to make them suitable for lung transplantation. Moderate hyperthermia is known to exert cytoprotective effect, via induction and accumulation of specific proteins, known as heat shock proteins (HSPs). We hypothesized that thermal preconditioning (TP) with a well-calibrated heat stress applied during EVLP could improve lung graft by reduction of oxidative stress, cell death and inflammation. <h3>Methods</h3> Male rats were randomly assigned into 5 groups: controls (Ctrl, n=5) and four heat stress groups (HS of 41.5°C; 42.0°C; 42.5°C or 43.5°C, n=5). In all groups, lungs were kept <i>in situ</i> for 1h at room temperature, then flushed with cold Perfadex. Heart-lung block was harvested and kept at 4°C for 1h, then mounted on the EVLP system for 3h. In Ctrl group, the temperature perfusion solution was kept at 37°C the all-time. In the HS groups, a heat shock was done for 30 minutes after 1h of EVLP by heating the perfusion solution at different temperature. At the end of EVLP, lungs were snap-frozen and kept at -80°C for further tissue analysis. <h3>Results</h3> Static pulmonary compliance SPC ratio (180 min to 60 min of EVLP) was significantly increased in the 41.5°C group compared to Ctrl group, while it was significantly reduced in 43.5°C group. The weight gain was significantly reduced in 41.5°C, whereas increased in 43.5°C group. In all HS groups, except in 43.5°C, protein levels of inducible HSP72 and Heme oxygenase-1 (HO-1) were significantly increased compared to Ctrl. Reduction of caspase 3 activation (apoptosis marker) was seen in all groups but reached significant level only in 41.5°C and 43.5°C groups. In all HS groups, we could see a reduction of inflammatory mediators such as, IL-1α, IL-1β, and TNF-α, a reduction of the release of CC16 (a marker of lung epithelial injury), as well as a reduction of IL-1β in Steen solution. <h3>Conclusion</h3> Thermal preconditioning during EVLP can decrease apoptotic cell death and inflammation of damaged lungs. These findings demonstrate that EVLP in association with well calibrated thermal preconditioning (41.5°C) could be a promising therapeutic platform improving lung quality of damaged donor lungs.

Lung Function and Inflammatory Profiling of Damaged Rat Donor Lungs Following EVLP Thermal Preconditioning

<h3>Purpose</h3> Ex-vivo lung perfusion (EVLP) is a validated technique allowing evaluation and reconditioning of damaged donor lungs to make them suitable for transplantation. Moderate hyperthermia is known to exert cytoprotective effect, via induction and accumulation of specific proteins, known as heat shock proteins (HSPs). We hypothesized that thermal preconditioning (TP) with a well-calibrated heat stress applied during EVLP could improve lung graft by reduction of oxidative stress, cell death and inflammation. <h3>Methods</h3> Male rats were randomly assigned into 2 groups: controls (Ctrl, n=5) or TP (n=5). In both groups, lungs were kept <i>in situ</i> for 1h at room temperature, then flushed with cold Perfadex. Heart-lung block was harvested and kept at 4°C for 1h, then mounted on the EVLP system for 3h. In Ctrl group, the temperature perfusion solution was kept at 37°C the all-time. In the TP group, a heat shock was done for 30 minutes after 1h of EVLP by heating the perfusion solution at 41.5°C. At the end of EVLP, lungs were snap-frozen and kept at -80°C for further tissue analysis. <h3>Results</h3> Static pulmonary compliance, peak inspiratory pressure, and oxygenation capacity were similar in both groups. We were able to induce HSPs in the TP group (<i>Hspa1a</i> and <i>Hmox-1</i>). In the TP group, we observed a reduction of oxidative stress (carbonyls and MDA), a decrease of apoptosis (Caspase 3 and 7) and a decrease of pro-inflammatory NF-κB transcription factor as well as the expression of inflammation mediators such as TNF-α, IL-1α, IL-1β, CXCL-1 and CCL-2. <h3>Conclusion</h3> Thermal preconditioning during EVLP decreased oxidative stress, apoptotic cell death, and inflammation of damaged lungs. These findings demonstrate that EVLP in association with thermal preconditioning could be a promising therapeutic platform improving lung quality of damaged donor lungs.

Effects of cold or warm ischemia and ex-vivo lung perfusion on the release of damage associated molecular patterns and inflammatory cytokines in experimental lung transplantation

Lung transplantation (LTx) is associated with sterile inflammation, possibly related to the release of damage associated molecular patterns (DAMPs) by injured allograft cells. We have measured cellular damage and the release of DAMPs and cytokines in an experimental model of LTx after cold or warm ischemia and examined the effect of pretreatment with ex-vivo lung perfusion (EVLP). Rat lungs were exposed to cold ischemia alone (CI group) or with 3h EVLP (CI-E group), warm ischemia alone (WI group) or with 3 hour EVLP (WI-E group), followed by LTx (2 hour). Bronchoalveolar lavage (BAL) was performed before (right lung) or after (left lung) LTx to measure LDH (marker of cellular injury), the DAMPs HMGB1, IL-33, HSP-70 and S100A8, and the cytokines IL-1β, IL-6, TNFα, and CXCL-1. Graft oxygenation capacity and static compliance after LTx were also determined. Compared to CI, WI displayed cellular damage and inflammation without any increase of DAMPs after ischemia alone, but with a significant increase of HMGB1 and functional impairment after LTx. EVLP promoted significant inflammation in both cold (CI-E) and warm (WI-E) groups, which was not associated with cell death or DAMP release at the end of EVLP, but with the release of S100A8 after LTx. EVLP reduced graft damage and dysfunction in warm ischemic, but not cold ischemic, lungs. The pathomechanisms of sterile lung inflammation during LTx are significantly dependent on the conditions. The release of HMGB1 (in the absence of EVLP) and S100A8 (following EVLP) may be important factors in the pathogenesis of LTx.

Alterations in Perfusate Leukocyte Populations are Associated with Donor Mode of Death and the Outcome of Ex Vivo Lung Perfusion

<h3>Purpose</h3> Lung transplantation (LTx) is limited by the scarcity of suitable donor lungs. One option to expand the donor pool is the use of donation after circulatory death (DCD) lungs. Additionally, ex vivo lung perfusion (EVLP) allows for evaluation and treatment of donor lungs prior to LTx. There are limited data describing the cellular, specifically leukocyte populations in EVLP perfusate, and their link to donor mode of death. We hypothesized that EVLP perfusate leukocytes differ between DCD and neurological determination of death (NDD) lungs, and between lungs accepted and declined for transplant. <h3>Methods</h3> EVLP perfusates were sampled hourly from clinical EVLP cases until termination. Perfusate cells were separated and cryopreserved. The first and last available perfusate from lungs accepted (n = 10 NDD and n = 10 DCD) and declined (n = 10 NDD and n = 10 DCD) for LTx were selected and stained with flow cytometry panels to characterize major leukocyte populations. <h3>Results</h3> Lymphoid cells form the majority of perfusate cells (Fig A). B cells were significantly higher in declined lungs regardless of mode of donor death (Fig B). CD4+ memory T cells (CD3+ CD4+ CD45RO+) and activated CD8+ T cells (CD3+ CD8+ CD25+) were elevated in the DCD lung perfusates (Fig C). In all groups, CD163hi, HLA-DR+ monocytes were observed at first available perfusate and diminished at the end of EVLP, while neutrophils were low in the first perfusate sample but increased significantly by the end of EVLP (Fig D). <h3>Conclusion</h3> Interestingly, leukocyte populations in EVLP perfusate differ between mode of donor death, with lower levels of B cells associated with lungs accepted for transplant, and certain T cell subsets over-represented in DCD lungs. While no significant changes were observed in myeloid cells, their kinetics over time vary based on cell type. Future studies will be required to determine the sources of these cell populations and whether they are involved in the pathogenesis of lung injury prior to transplant.

Donor Neutrophils and B Cells in EVLP Perfusate are Associated with Severe Primary Graft Dysfunction

<h3>Purpose</h3> Primary graft dysfunction (PGD) remains a major obstacle to lung transplantation (LT) success and is classified as severe (PGD3) in 10-15% of all LT recipients. Ex vivo lung perfusion (EVLP) allows for evaluation of donor lungs prior to LT and measurement of circulating cells and soluble factors in the perfusate. The contributions of donor cells to PGD are unknown, and whether specific cells might confer a risk of PGD is unclear. We hypothesized that larger quantities of donor myeloid cells in EVLP perfusate might predict PGD3. <h3>Methods</h3> In this retrospective study, we selected consecutive clinical EVLP cases and grouped them based on Pa_O2/F_IO2 (P/F) ratio at 72h post-LT, resulting in two groups: PGD3 (n = 33, P/F = 183 ± 48mmHg) and PGD0/1 (n = 25, P/F = 395 ± 71mmHg). Cryopreserved perfusate cells from the first and last hour of EVLP were subjected to multiparameter flow cytometry to identify major leukocyte populations. Time and PGD-dependent differences were assessed by two-way ANOVA with post-hoc testing adjusted for multiple comparisons. <h3>Results</h3> In the first hour of EVLP, the total cells/mL was higher in the PGD3 group compared to PGD0/1 (12.1 × 10⁵ versus 8.4 × 10⁵ cells/ml, p = 0.0573) - the cell/ml decreased in both groups to similar densities by the end of EVLP (Fig A). In the PGD3 group, neutrophils (Fig B) and B cells (Fig C) were significantly higher than in the PGD0/1 group in the first hour and significantly decreased over time. Macrophages (Fig D) and classical monocytes (Fig E) were also elevated in PGD3 group in the first hour, although this observation did not reach statistical significance. <h3>Conclusion</h3> Neutrophils and B cells in the 1-hour perfusate are associated with PGD3 development at 72h post-LT. While these findings suggest that higher numbers of these donor cells may play a role in PGD pathogenesis, PGD3 developed even though these populations were reduced by the end of EVLP. Studies of cellular activation states will be conducted to validate these findings and explore donor cellular contributions to PGD.

Prediction of donor related lung injury in clinical lung transplantation using a validated ex vivo lung perfusion inflammation score

Ex vivo lung perfusion (EVLP) is an isolated organ assessment technique that has revolutionized the field of lung transplantation and enabled a safe increase in the number of organs transplanted. The objective of this study was to develop a protein-based assay that would provide a precision medicine approach to lung injury assessment during EVLP. Perfusate samples collected from clinical EVLP cases performed from 2009 to 2019 were separated into development (n = 281) and validation (n = 57) sets to derive and validate an inflammation score based on IL-6 and IL-8 protein levels in perfusate. The ability of an inflammation score to predict lungs suitable for transplantation and likely to produce excellent recipient outcomes (time on ventilator ≤ 3 days) was assessed. Inflammation scores were compared to conventional clinical EVLP assessment parameters and associated with outcomes, including primary graft dysfunction and patient care in the ICU. An inflammation score accurately predicted the decision to transplant (AUROC 68% [95% CI 62-74]) at the end of EVLP and those transplants associated with short ventilator times (AUROC 73% [95% CI 66-80]). The score identified lungs more likely to develop primary graft dysfunction at 72-hours post-transplant (OR 4.0, p = 0.03). A model comprised of the inflammation score and ∆PO2 was able to determine EVLP transplants that were likely to have excellent recipient outcomes, with an accuracy of 87% [95% CI 83-92]. The adoption of an inflammation score will improve accuracy of EVLP decision-making and increase confidence of surgical teams to determine lungs that are suitable for transplantation, thereby improving organ utilization rates and patient outcomes.

HSP90 Inhibitor Improves Lung Protection in Porcine Model of Donation After Circulatory Arrest

Ischemia-reperfusion associated with prolonged warm ischemia during donation after circulatory death (DCD) induces acute lung injury. The objective of this study was to combine exvivo lung perfusion (EVLP) and a heat shock protein-90 inhibitor (HSP90i) to recondition DCD organs and prevent primary graft dysfunction. Pigs (55 to 65 kg) were anesthetized, ventilated, and hemodynamically monitored. Cardiac arrest was induced with potassium chloride, and animals were left nonventilated for 2 hours. Lungs were procured and perfused in an EVLP platform for 4 hours by using a cellular perfusate. In the study group, the perfusate contained HSP90i and its transport vehicle (n= 4). In the control group, the perfusate contained only the transport vehicle (n= 4). Gas exchange, airway pressures, and compliance were measured. Pulmonary edema was assessed by bronchoscopy and weight measurement. Lung biopsy samples were obtained for histologic analyses and protein expression measurements. The use of HSP90i reduced lung weight gain to 8.4 ± 3.4% vs 26.6 ± 6.2% in the control group (P < .05). There was reduced edema formation. The ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen at the end of EVLP was 423 ± 65 in the study group vs 339 ± 25 mm Hg in the control group, but this difference was not statistically significant. Lactate metabolism, pulmonary vascular resistance, and pulmonary arterial pressure improved during EVLP with the use of the HSP90i. The use of HSP90i with EVLP improves the lung reconditioning process. Further research is required to confirm whether these findings translate to benefit once transplanted and observed invivo. Successful pharmacologic inhibitors may expand the donor pool in the context of DCD donors.

Alterations in Perfusate Leukocyte Populations are Associated with Donor Mode of Death and the Outcome of Ex Vivo Lung Perfusion

Lung transplantation (LTx) is limited by the scarcity of suitable donor lungs. One option to expand the donor pool is the use of donation after circulatory death (DCD) lungs. Additionally, ex vivo lung perfusion (EVLP) allows for evaluation and treatment of donor lungs prior to LTx. There are limited data describing the cellular, specifically leukocyte populations in EVLP perfusate, and their link to donor mode of death. We hypothesized that EVLP perfusate leukocytes differ between DCD and neurological determination of death (NDD) lungs, and between lungs accepted and declined for transplant. EVLP perfusates were sampled hourly from clinical EVLP cases until termination. Perfusate cells were separated and cryopreserved. The first and last available perfusate from lungs accepted (n = 10 NDD and n = 10 DCD) and declined (n = 10 NDD and n = 10 DCD) for LTx were selected and stained with flow cytometry panels to characterize major leukocyte populations. Lymphoid cells form the majority of perfusate cells (Fig A). B cells were significantly higher in declined lungs regardless of mode of donor death (Fig B). CD4+ memory T cells (CD3+ CD4+ CD45RO+) and activated CD8+ T cells (CD3+ CD8+ CD25+) were elevated in the DCD lung perfusates (Fig C). In all groups, CD163hi, HLA-DR+ monocytes were observed at first available perfusate and diminished at the end of EVLP, while neutrophils were low in the first perfusate sample but increased significantly by the end of EVLP (Fig D). Interestingly, leukocyte populations in EVLP perfusate differ between mode of donor death, with lower levels of B cells associated with lungs accepted for transplant, and certain T cell subsets over-represented in DCD lungs. While no significant changes were observed in myeloid cells, their kinetics over time vary based on cell type. Future studies will be required to determine the sources of these cell populations and whether they are involved in the pathogenesis of lung injury prior to transplant.

Real-Time Lung Weight Measurement: A Simple Predictor for Clinical Outcomes of Ex-Vivo Lung Perfusion

Lung weight (LW) gain is a typical characteristic of ischemia reperfusion injury in ex-vivo lung perfusion (EVLP). However, LW gain data is available only at the end of EVLP. Additionally, objective physiological parameters useful in EVLP evaluation are limited. The study purpose was to investigate the usefulness of real-time LW measurement to assess pulmonary edema and to predict clinical outcomes. LW at back table was measured before and after EVLP. During acellular EVLP, real-time LW was continuously measured by a scale attached under the organ chamber. The LW change (weight change per time, g/min) was measured during the non-touching period at multiple time points and the estimated LW gain was calculated by LW change and its duration. Transplant suitability was judged based on the standard criteria including trend of physiological parameters and palpation findings. Ten lungs were perfused at our institute and 8 lungs were clinically transplanted. All patients survived and primary graft dysfunction (PGD) grade at 72 hours were Grade 0-1, n = 6; Grade 2, n = 2; Grade 3, n = 0. Estimated LW gain during the entire perfusion period significantly correlated with LW gain at the back table (n = 10, r = 0.95, p < 0.01, Fig. 1A). Furthermore, initial LW gain (0-60 min) had a significant correlation with back table LW gain, compared with LW gain after 60 min of EVLP (n = 10, r = 0.79, p < 0.01, Fig. 1B). Non-suitable group had significantly higher initial LW gain compared to suitable group (120 ± 30 vs -17 ± 56 g, p = 0.02, Fig. 1C). Unfavorable outcome group (defined as non-suitable or PGD grade 2-3) group had significantly higher initial LW gain compared to favorable outcome group (defined as PGD grade 0-1, 75 ± 56 vs -32 ± 58 g, p = 0.02, Fig. 1D). The data suggest that LW gain calculated by real-time LW measurement might be useful to assess pulmonary edema during EVLP. Initial LW gain at 0-60 min of EVLP might be a predictor of transplant suitability and clinical outcomes.

Assessment of Donor Lung Aspiration on the Ex Vivo Lung Perfusion (EVLP) Platform

Donor aspiration of gastric contents represents a significant risk for lung injury and severe primary graft dysfunction after lung transplantation. Concern about possible aspiration is a common reason for clinicians to decline donor lungs for transplantation. The presence of bile acid in donor bronchial wash (BW) or bronchoalveolar lavage (BAL) fluid may indicate that a donor has aspirated. Previous reports have also shown that aspiration of bile acids cause increased inflammation in the lung. Ex vivo lung perfusion (EVLP) has become an important platform to assess questionable or "marginal" donors. In this study, we examined the relationship between possible donor aspiration and physiologic and biologic assessment of inflammation on EVLP. Large airway BW samples were collected from 270 donors (n=111 non-EVLP, n=159 EVLP) prior to transplantation. Bile acid levels were measured in BW samples using a commercially available colorimetric assay and clinically approved platform. Protein and biochemical measurements were made in perfusate samples collected hourly after the start of EVLP. The majority of donor lungs with clinically suspected aspiration were successfully transplanted after EVLP assessment (58% [23/40]). Donor bile acid levels were significantly higher in donor lungs that were ultimately declined for transplantation at the end of EVLP (0.28 vs. 0.12 μM, p<0.05, n=143). During EVLP there was a positive correlation between bile acid in the donor BW and inflammation (IL-8 levels (r=0.19, p<0.05, n=159)). Similarly, there was an increase in biochemical markers of cell death ([Ca2+]) in the EVLP perfusate of donors with high bile acid levels in BW (r=0.27, p<0.001, n=142). Following EVLP and transplantation, we observed no significant effects of donor bile acid levels on recipient ICU length-of-stay (r=0.09, p=0.35, n=117). Suspected donor aspiration can be assessed using the EVLP platform. Bile acid levels measured in the donor lung are associated with increased inflammation and lung injury observed during EVLP, but not recipient outcomes post-EVLP. Therefore, EVLP may be an ideal platform to identify marginal donor lungs suspected of aspiration but are ultimately suitable for clinical transplantation.

Donor Leukocyte Trafficking and Damage-associated Molecular Pattern Expression During Ex Vivo Lung Perfusion.

Background.While ex vivo lung perfusion (EVLP) has become established in lung transplantation, the cellular processes occurring during this period are not yet fully understood. Prior studies demonstrated that donor leukocytes (DLs) migrate from the graft into the perfusate during EVLP, but the distribution of DLs in graft and perfusate compartments has not been characterized. Moreover, cell death of DLs has been implicated in mediating graft injury during EVLP, but the underlying mechanisms have not been elucidated. We hypothesized the following: (1) there is a nonspecific migration of DLs from the graft into perfusate and (2) cell death of DLs releases damage-associated molecular patterns (DAMPs) that contribute to the inflammatory milieu during EVLP.Methods.EVLP was performed on rat lungs for 3 hours (N = 6). At the end of EVLP, flow cytometry was used to quantify the distribution of different DL cell types in both the graft and perfusate compartments. During EVLP, the perfusate was also sampled hourly to measure levels of DAMPs and downstream inflammatory cytokines generated during EVLP.Results.At the conclusion of EVLP, there was a significantly higher proportion of T and B cells present in the perfusate compartment compared with the graft compartment. There was a time-dependent increase in extracellular DNA and tumor necrosis factor α in the perfusate during EVLP.Conclusions.T cells and B cells are enriched in the perfusate compartment during EVLP. Cell death of DLs contributes to an accumulation of DAMPs during EVLP.

Increased Arginase Expression and Decreased Nitric Oxide in Pig Donor Lungs after Normothermic Ex Vivo Lung Perfusion.

An established pig lung transplantation model was used to study the effects of cold ischemia time, normothermic acellular ex vivo lung perfusion (EVLP) and reperfusion after lung transplantation on l-arginine/NO metabolism in lung tissue. Lung tissue homogenates were analyzed for NO metabolite (NOx) concentrations by chemiluminescent NO-analyzer technique, and l-arginine, l-ornithine, l-citrulline and asymmetric dimethylarginine (ADMA) quantified using liquid chromatography-mass spectrometry (LC-MS/MS). The expression of arginase and nitric oxide synthase (NOS) isoforms in lung was measured by real-time polymerase chain reaction. EVLP preservation resulted in a significant decrease in concentrations of NOx and l-citrulline, both products of NOS, at the end of EVLP and after reperfusion following transplantation, compared to control, respectively. The ratio of l-ornithine over l-citrulline, a marker of the balance between l-arginine metabolizing enzymes, was increased in the EVLP group prior to reperfusion. The expression of both arginase isoforms was increased from baseline 1 h post reperfusion in EVLP but not in the no-EVLP group. These data suggest that EVLP results in a shift of the l-arginine balance towards arginase, leading to NO deficiency in the lung. The arginase/NOS balance may, therefore, represent a therapeutic target to improve lung quality during EVLP and, subsequently, transplant outcomes.

Impact of imipenem concentration in lung perfusate and tissue biopsy during clinical ex-vivo lung perfusion of high-risk lung donors.

Normothermic ex-vivo lung perfusion (EVLP) limits organ donor shortage by potentially using high-risk donor lungs. Microbial burden reduction has been demonstrated after EVLP using antibiotic prophylaxis with imipenem. However, no data have been published on the clinical consequences of the potential residual bacterial burden. Imipenem concentration was measured every hour (T0 to T6) in the lung perfusate and at the end of EVLP (Tf) in biopsies. The antimicrobial activity of perfusate at T1 and Tf against E. coli and K. pneumoniae was evaluated. Lungs were distinguished: no bacterial species in recipients and donors (donor-/recipient-); bacterial species isolated from donors and not from recipients (donor+/recipient-); same bacterial species in both recipients and donors (donor+/recipient+). Interleukin 6 (IL-6) and IL-8 concentrations in lung perfusate, clinical pulmonary infection score (CPIS) and primary graft dysfunction (PGD) were evaluated. Imipenem concentration in perfusate decreased over time. T1 and Tf perfusates exhibited bactericidal activity against E. coli and K. pneumoniae. Overall, T1 perfusates yielded higher bactericidal titers (BTs) than Tf. The donor+/recipient+ group (26% of cases) had higher IL-6 and IL-8 in perfusate and higher CPIS. Recipients with the same bacterial species isolated in their donors had higher risk of pulmonary inflammation and early post-transplant pneumonia. Improvements in antimicrobial strategies during EVLP are warranted to minimize the consequences of donor associated respiratory infection.

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.

Assessment of lung edema during ex-vivo lung perfusion by single transpulmonary thermodilution: A preliminary study in humans.

Single transpulmonary thermodilution (SD) with extravascular lung water index (EVLWI) could become a new tool to better assess lung graft edema during ex-vivo lung perfusion (EVLP). In this study we compare EVLWI with conventional methods to better select lungs during EVLP and to predict post-transplant primary graft dysfunction (PGD). We measured EVLWI, arterial oxygen/fraction of inspired oxygen (P/F) ratio, and static lung compliance (SLC) during EVLP in an observational study. At the end of EVLP, grafts were accepted or rejected according to a standardized protocol blinded to EVLWI results. We compared the respective ability of EVLWI, P/F, and SLC to predict PGD. Mann-Whitney U-test, Fisher's exact test, and receiver-operating characteristic (ROC) curve data were used for analysis. p < 0.05 was considered statistically significant. Thirty-five lungs were evaluated by SD during EVLP. Three lungs were rejected for pulmonary edema. Thirty-two patients were transplanted, 8 patients developed Grade 2 or 3 PGD, and 24 patients developed Grade 0 or 1 PGD. In contrast to P/F ratio, SLC, and pulmonary artery pressure, EVLWI differed between these 2 populations (p < 0.001). The area under the ROC for EVLWI assessing Grade 2 or 3 PGD at the end of EVLP was 0.93. Donor lungs with EVLWI >7.5 ml/kg were more likely associated with a higher incidence of Grade 2 or 3 PGD at Day 3. Increased EVLWI during EVLP was associated with PGD in recipients.

Ex vivo administration of trimetazidine improves post-transplant lung function in pig model.

Ex vivo lung perfusion (EVLP) is not only used to assess marginal donor lungs but is also used as a platform to deliver therapeutic agents outside the body. We previously showed the beneficial effects of trimetazidine (TMZ) on ischaemia reperfusion (IR) injury in a rat model. This study evaluated the effects of TMZ in a pig EVLP transplant model. Pig lungs were retrieved and stored for 24 h at 4°C, followed by 4 h of EVLP. Allografts were randomly allocated to 2 groups ( n = 5 each). TMZ (5 mg/kg) was added to the prime solution prior to EVLP. After EVLP, left lungs were transplanted and recipients were observed for 4 h. Allograft gas exchange function and lung mechanics were recorded hourly throughout reperfusion. Microscopic lung injury and inflammatory and biochemical parameters were assessed. There was a trend towards better oxygenation during EVLP in the TMZ group ( P = 0.06). After transplantation, pulmonary gas exchange was significantly better during the 4-h reperfusion period and after isolation of the allografts for 10 min ( P < 0.05). Tissue thiobarbituric acid levels, myeloperoxidase activity and protein concentrations in bronchoalveolar lavage samples were significantly lower in the TMZ group at the end of EVLP ( P < 0.05). Ex vivo treatment of donor lungs with TMZ significantly improved immediate post-transplant lung function. Further studies are warranted to understand the effect of this strategy on long-term lung function.

A porcine ex vivo lung perfusion model with maximal argon exposure to attenuate ischemia-reperfusion injury.

Argon (Ar) is a noble gas with known organoprotective effects in rodents and in vitro models. In a previous study we failed to find a postconditioning effect of Ar during ex vivo lung perfusion (EVLP) on warm-ischemic injury in a porcine model. In this study, we further investigated a prolonged exposure to Ar to decrease cold ischemia-reperfusion injury after lung transplantation in a porcine model with EVLP assessment. Domestic pigs (n = 6/group) were pre-conditioned for 6 hours with 21% O2 and 79% N2 (CONTR) or 79% Ar (ARG). Subsequently, lungs were cold flushed and stored inflated on ice for 18 hours inflated with the same gas mixtures. Next, lungs were perfused for 4 hours on EVLP (acellular) while ventilated with 12% O2 and 88% N2 (CONTR group) or 88% Ar (ARG group). The perfusate was saturated with the same gas mixture but with the addition of CO2 to an end-tidal CO2 of 35-45 mmHg. The saturated perfusate was drained and lungs were perfused with whole blood for an additional 2 hours on EVLP. Evaluation at the end of EVLP did not show significant effects on physiologic parameters by prolonged exposure to Ar. Also wet-to-dry weight ratio did not improve in the ARG group. Although in other organ systems protective effects of Ar have been shown, we did not detect beneficial effects of a high concentration of Ar on cold pulmonary ischemia-reperfusion injury in a porcine lung model after prolonged exposure to Ar in this porcine model with EVLP assessment.

Bronchodilator Inhalation During Ex Vivo Lung Perfusion Improves Posttransplant Graft Function After Warm Ischemia

We hypothesized that an injured lung graft from donation after cardiac death donors could be reconditioned before transplantation using an exvivo lung perfusion (EVLP) system and ventilation with high-dose short-acting β2-adrenergic receptor agonists. Cardiac arrest was induced in a canine model by intravenous potassium chloride injection. Lungs were randomly assigned to two groups after 150 minutes of warm ischemia: inhalation of 1,400 μg of procaterol (BETA group, n= 5) or control group receiving solvent (CON group, n= 5) during EVLP. Left lungs were transplanted after 120 minutes of EVLP. Functional variables, tissue adenosine 5'-triphosphate levels, and tissue cyclic adenosine monophosphate levels were measured 240 minutes after transplantation. Physiologic pulmonary function was similar at the end of EVLP in both groups. However, significantly better graft oxygenation, dynamic pulmonary compliance, and reduced pulmonary vascular resistance were observed in the BETA group than in the CON group 240 minutes after transplantation. No severe adverse effects were observed after lung transplantation in the BETA group. Lung tissue adenosine 5'-triphosphate levels and cyclic adenosine monophosphate levels were significantly higher in the BETA group than in the CON group at the end of EVLP and at 240 minutes after transplantation. High-dose nebulized procaterol during EVLP ameliorated lung graft dysfunction at the early posttransplantation period without severe adverse effects. These data suggest that lung reconditioning with procaterol ventilation during EVLP improves lung graft function after transplantation.

Experimental Ex Vivo Lung Perfusion with Sevoflurane: Effect on donors after circulatory death (DCD) lung grafts in a rodent model.

Ischemia-reperfusion injury is a key mechanism of graft damage during lung transplantation, which could be targeted by therapies applied during ex-vivo lung perfusion (EVLP). The inhalational anaesthetic Sevoflurane was found to protect to some degree against ischemia-reperfusion injury1. In this experimental study we wanted to determine the therapeutic potential of volatile Sevoflurane added to the perfusate during EVLP of damaged lung grafts. Two groups of 6 Sprague-Dawley rats were used. After cardiac arrest and a warm ischemic time of 1 hour the lungs were flushed with cold Perfadex®, harvested and kept for 2 hours at 4°C. Normothermic EVLP during 3 hours was performed using a customized circuit primed either with Steen solution® only (control group) or supplemented within the first 30 minutes of EVLP with a gas mixture containing 2% of sevoflurane (treatment group). Differential oxygen partial pressures in the perfusate (ΔvapO2), pulmonary artery pressure (PAP), vascular resistance (PVR), lung compliance (LC), peak airway pressure (PAWP) and lung weight gain (WG) were measured. At the end of EVLP, protein and lactate dehydrogenase (LDH) levels were determined in bronchoalveolar lavage (BAL) and interleukin-6 (IL-6) and protein carbonyl (index of oxidative stress) were measured in lung tissue. Normothermic EVLP of lungs harvested after warm ischemia resulted in markedly declined ΔvapO2, LC, and elevated PAP, PVR, PAWP, WG. In contrast, lungs treated with Sevoflurane during EVLP displayed significantly improved LC, reduced weight gain and significantly lower LDH and protein carbonyl levels as compared to controls. PAWP and IL-6 were diminished in treated lungs, but were not statistically different to controls. The intravascular administration of sevoflurane during EVLP reduces oxidative stress and improves the functional status of DCD rat lungs in this experimental setting.

Ex Vivo Lung Perfusion of Cardiac-death Donor Lung in Pigs

Background: Lung transplantation (LTx) is a life-saving treatment for patients with end-stage lung disease; however, the shortage of donor lungs has been a major limiting factor to increasing the number of LTx. Growing experience following LTx using donor lungs after cardiac death (DCD) has been promising, although concerns remain. The purpose of this study was to develop a DCD lung harvest model using an ex vivo lung perfusion (EVLP) system and to assess the function of presumably damaged lungs harvested from the DCD donor in pigs. Methods: The 40 kg pigs were randomly divided into the control group with no ischemic lung injury (n=5) and the study group (n=5), which had 1 hour of warm ischemic lung injury after cardiac arrest. Harvested lungs were placed in the EVLP circuit and oxygen capacities (OC), pulmonary vascular resistance (PVR), and peak airway pressure (PAP) were evaluated every hour for 4 hours. At the end of EVLP, specimens were excised for pathologic review and wet/dry ratio. Results: No statistically significant difference in OC (P=0.353), PVR (P=0.951), and PAP (P=0.651) was observed in both groups. Lung injury severity score (control group vs. study group: 0.700±0.303 vs. 0.870±0.130; P=0.230) and wet/dry ratio (control group vs. study group: 5.89±0.97 vs. 6.20±0.57; P=0.560) also showed no statistically significant difference between the groups. Conclusions: The function of DCD lungs assessed using EVLP showed no difference from that of control lungs without ischemic injury; therefore, utilization of DCD lungs can be a new option to decrease the number of deaths on the waiting list.