Proteomic Analysis of Primary Graft Dysfunction in Porcine Lung Transplantation Reveals Alveolar-Capillary Barrier Changes Underlying the High Particle Flow Rate in Exhaled Breath.

Primary graft dysfunction (PGD) remains a challenge for lung transplantation (LTx) recipients as a leading cause of poor early outcomes. New methods are needed for more detailed monitoring and understanding of the pathophysiology of PGD. The measurement of particle flow rate (PFR) in exhaled breath is a novel tool to monitor and understand the disease at the proteomic level. In total, 22 recipient pigs underwent orthotopic left LTx and were evaluated for PGD on postoperative day 3. Exhaled breath particles (EBPs) were evaluated by mass spectrometry and the proteome was compared to tissue biopsies and bronchoalveolar lavage fluid (BALF). Findings were confirmed in EBPs from 11 human transplant recipients. Recipients with PGD had significantly higher PFR [686.4 (449.7-8,824.0) particles per minute (ppm)] compared to recipients without PGD [116.6 (79.7-307.4) ppm, p = 0.0005]. Porcine and human EBP proteins recapitulated proteins found in the BAL, demonstrating its utility instead of more invasive techniques. Furthermore, adherens and tight junction proteins were underexpressed in PGD tissue. Histological and proteomic analysis found significant changes to the alveolar-capillary barrier explaining the high PFR in PGD. Exhaled breath measurement is proposed as a rapid and non-invasive bedside measurement of PGD.

High resolution fluorescence imaging of the alveolar scaffold as a novel tool to assess lung injury.

Acute lung injury (ALI) represents an aetiologically diverse form of pulmonary damage. Part of the assessment and diagnosis of ALI depends on skilled observer-based scoring of brightfield microscopy tissue sections. Although this readout is sufficient to determine gross alterations in tissue structure, its categorical scores lack the sensitivity to describe more subtle changes in lung morphology. To generate a more sensitive readout of alveolar perturbation we carried out high resolution immunofluorescence imaging on 200 µm lung vibratome sections from baseline and acutely injured porcine lung tissue, stained with a tomato lectin, Lycopersicon Esculentum Dylight-488. With the ability to resolve individual alveoli along with their inner and outer wall we generated continuous readouts of alveolar wall thickness and circularity. From 212 alveoli traced from 10 baseline lung samples we established normal distributions for alveolar wall thickness (27.37; 95% CI [26.48:28.26]) and circularity (0.8609; 95% CI [0.8482:0.8667]) in healthy tissue. Compared to acutely injured lung tissue baseline tissue exhibited a significantly lower wall thickness (26.86 ± 0.4998 vs 50.55 ± 4.468; p = 0.0003) and higher degree of circularityϕ≤ (0.8783 ± 0.01965 vs 0.4133 ± 0.04366; p < 0.0001). These two components were subsequently combined into a single more sensitive variable, termed the morphological quotient (MQ), which exhibited a significant negative correlation (R2 = 0.9919, p < 0.0001) with the gold standard of observer-based scoring. Through the utilisation of advanced light imaging we show it is possible to generate sensitive continuous datasets describing fundamental morphological changes that arise in acute lung injury. These data represent valuable new analytical tools that can be used to precisely benchmark changes in alveolar morphology both in disease/injury as well as in response to treatment/therapy.

Design and Rationale of Cytokine Filtration in Lung Transplantation (GLUSorb): Protocol for a Multicenter Clinical Randomized Controlled Trial.

BACKGROUND: Lung transplantation (LTx) is the only treatment option for end-stage lung disease. Despite improvements, primary graft dysfunction (PGD) remains the leading cause of early mortality and precipitates chronic lung allograft dysfunction, the main factor in late mortality after LTx. PGD develops within the first 72 hours and impairs the oxygenation capacity of the lung, measured as partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2). Increasing the PaO2/FiO2 ratio is thus critical and has an impact on survival. There is a general lack of effective treatments for PGD. When a transplanted lung is not accepted by the immune system in the recipient, a systemic inflammatory response starts where cytokines play a critical role in initiating, amplifying, and maintaining the inflammation leading to PGD. Cytokine filtration can remove these cytokines from the circulation, thus reducing inflammation. In a proof-of-concept preclinical porcine model of LTx, cytokine filtration improved oxygenation and decreased PGD. In a feasibility study, we successfully treated patients undergoing LTx with cytokine filtration (ClinicalTrials.gov; NCT05242289). OBJECTIVE: The purpose of this clinical trial is to demonstrate the superiority of cytokine filtration in improving LTx outcome, based on its effects on oxygenation ratio, plasma levels of inflammatory markers, PGD incidence and severity, lung function, kidney function, survival, and quality of life compared with standard treatment with no cytokine filtration. METHODS: This study is a Swedish national interventional randomized controlled trial involving 116 patients. Its primary objective is to investigate the potential benefits of cytokine filtration when used in conjunction with LTx. Specifically, this study aims to determine whether the application of cytokine filtration, administered for a duration of 12 hours within the initial 24 hours following a LTx procedure, can lead to improved patient outcomes. This study seeks to assess various aspects of patient recovery and overall health to ascertain the potential positive impact of this intervention on the posttransplantation course. RESULTS: The process of patient recruitment for this study is scheduled to commence subsequent to a site initiation visit, which was slated to take place on August 28, 2023. The primary outcome measure that will be assessed in this research endeavor is the oxygenation ratio, a metric denoted as the highest PaO2/FiO2 ratio achieved by patients within a 72-hour timeframe following their LTx procedure. CONCLUSIONS: We propose that cytokine filtration could enhance the overall outcomes of LTx. Our hypothesis suggests potential improvements in LTx outcome and patient care. TRIAL REGISTRATION: ClinicalTrials.gov NCT05526950; https://www.clinicaltrials.gov/study/NCT05526950. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52553.

Proteomic changes to immune and inflammatory processes underlie lung preservation using ex vivo cytokine adsorption.

Introduction: In recent years, the field of graft preservation has made considerable strides in improving outcomes related to solid organ restoration and regeneration. Ex vivo lung perfusion (EVLP) in line with the related devices and treatments has yielded promising results within preclinical and clinical studies, with the potential to improve graft quality. Its main benefit is to render marginal and declined donor lungs suitable for transplantation, ultimately increasing the donor pool available for transplantation. In addition, using such therapies in machine perfusion could also increase preservation time, facilitating logistical planning. Cytokine adsorption has been demonstrated as a potentially safe and effective therapy when applied to the EVLP circuit and post-transplantation. However, the mechanism by which this therapy improves the donor lung on a molecular basis is not yet fully understood. Methods: We hypothesized that there were characteristic inflammatory and immunomodulatory differences between the lungs treated with and without cytokine adsorption, reflecting proteomic changes in the gene ontology pathways and across inflammation-related proteins. In this study, we investigate the molecular mechanisms and signaling pathways of how cytokine adsorption impacts lung function when used during EVLP and post-transplantation as hemoperfusion in a porcine model. Lung tissues during EVLP and post-lung transplantation were analyzed for their proteomic profiles using mass spectrometry. Results: We found through gene set enrichment analysis that the inflammatory and immune processes and coagulation pathways were significantly affected by the cytokine treatment after EVLP and transplantation. Conclusion: In conclusion, we showed that the molecular mechanisms are using a proteomic approach behind the previously reported effects of cytokine adsorption when compared to the non-treated transplant recipients undergoing EVLP.

Nothing but NETs: Cytokine adsorption correlates with lower circulating nucleosomes and is associated with decreased primary graft dysfunction.

Elevated levels of neutrophil extracellular traps (NETs) have been reported in primary graft dysfunction, making methods to reduce or remove them highly valuable. The mechanisms behind primary graft dysfunction (PGD) remain rudimentarily understood but its relation to higher rates of acute and chronic rejection necessitates the development of preventative treatments. This case series explores the use of a cytokine adsorber during lung transplantation with the focus of reducing circulating nucleosome levels as a measure of NETs. Treated patients showed reduced levels of circulating nucleosomes and remained free from PGD and histopathological signs of acute rejection at 1- and 3-month post-transplant. In contrast, patients without the adsorber experienced higher levels of circulating nucleosomes, PGD grades 1 and 3, and histopathological signs of acute rejection. Using a cytokine adsorber during transplantation may provide a reduced systemic inflammatory state with lower levels of NETs and consequently support graft acceptance.

Integrin α10ß1-selected mesenchymal stem cells reduced hypercoagulopathy in a porcine model of acute respiratory distress syndrome.

Mesenchymal stem cells (MSCs) have been studied for their potential benefits in treating acute respiratory distress syndrome (ARDS) and have reported mild effects when trialed within human clinical trials. MSCs have been investigated in preclinical models with efficacy when administered at the time of lung injury. Human integrin α10ß1-selected adipose tissue-derived MSCs (integrin α10ß1-MSCs) have shown immunomodulatory and regenerative effects in various disease models. We hypothesized that integrin α10ß1 selected-MSCs can be used to treat a sepsis-induced ARDS in a porcine model when administering cells after established injury rather than simultaneously. This was hypothesized to reflect a clinical picture of treatment with MSCs in human ARDS. 12 pigs were randomized to the treated or placebo-controlled group prior to the induction of mild to moderate ARDS via lipopolysaccharide administration. The treated group received 5 × 106 cells/kg integrin α10ß1-selected MSCs and both groups were followed for 12 h. ARDS was confirmed with blood gases and retrospectively with histological changes. After intervention, the treated group showed decreased need for inotropic support, fewer signs of histopathological lung injury including less alveolar wall thickening and reduction of the hypercoagulative disease state. The MSC treatment was not associated with adverse events over the monitoring period. This provides new opportunities to investigate integrin α10ß1-selected MSCs as a treatment for a disease which does not yet have any definitive therapeutic options.

Releasing high positive end-expiratory pressure to a low level generates a pronounced increase in particle flow from the airways.

OBJECTIVES: Detecting particle flow from the airways by a non-invasive analyzing technique might serve as an additional tool to monitor mechanical ventilation. In the present study, we used a customized particles in exhaled air (PExA) technique, which is an optical particle counter for the monitoring of particle flow in exhaled air. We studied particle flow while increasing and releasing positive end-expiratory pressure (PEEP). The aim of this study was to investigate the impact of different levels of PEEP on particle flow in exhaled air in an experimental setting. We hypothesized that gradually increasing PEEP will reduce the particle flow from the airways and releasing PEEP from a high level to a low level will result in increased particle flow. METHODS: Five fully anesthetized domestic pigs received a gradual increase of PEEP from 5 cmH2O to a maximum of 25 cmH2O during volume-controlled ventilation. The particle count along with vital parameters and ventilator settings were collected continuously and measurements were taken after every increase in PEEP. The particle sizes measured were between 0.41 µm and 4.55 µm. RESULTS: A significant increase in particle count was seen going from all levels of PEEP to release of PEEP. At a PEEP level of 15 cmH2O, there was a median particle count of 282 (154-710) compared to release of PEEP to a level of 5 cmH2O which led to a median particle count of 3754 (2437-10,606) (p < 0.009). A decrease in blood pressure was seen from baseline to all levels of PEEP and significantly so at a PEEP level of 20 cmH2O. CONCLUSIONS: In the present study, a significant increase in particle count was seen on releasing PEEP back to baseline compared to all levels of PEEP, while no changes were seen when gradually increasing PEEP. These findings further explore the significance of changes in particle flow and their part in pathophysiological processes within the lung.

A Semi-quantitative Scoring System for Green Histopathological Evaluation of Large Animal Models of Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is a life-threatening, high mortality pulmonary condition characterized by acute lung injury (ALI) resulting in diffuse alveolar damage. Despite progress regarding the understanding of ARDS pathophysiology, there are presently no effective pharmacotherapies. Due to the complexity and multiorgan involvement typically associated with ARDS, animal models remain the most commonly used research tool for investigating potential new therapies. Experimental models of ALI/ARDS use different methods of injury to acutely induce lung damage in both small and large animals. These models have historically played an important role in the development of new clinical interventions, such as fluid therapy and the use of supportive mechanical ventilation (MV). However, failures in recent clinical trials have highlighted the potential inadequacy of small animal models due to major anatomical and physiological differences, as well as technical challenges associated with the use of clinical co-interventions [e.g., MV and extracorporeal membrane oxygenation (ECMO)]. Thus, there is a need for larger animal models of ALI/ARDS, to allow the incorporation of clinically relevant measurements and co-interventions, hopefully leading to improved rates of clinical translation. However, one of the main challenges in using large animal models of preclinical research is that fewer species-specific experimental tools and metrics are available for evaluating the extent of lung injury, as compared to rodent models. One of the most relevant indicators of ALI in all animal models is evidence of histological tissue damage, and while histological scoring systems exist for small animal models, these cannot frequently be readily applied to large animal models. Histological injury in these models differs due to the type and severity of the injury being modeled. Additionally, the incorporation of other clinical support devices such as MV and ECMO in large animal models can lead to further lung damage and appearance of features absent in the small animal models. Therefore, semi-quantitative histological scoring systems designed to evaluate tissue-level injury in large animal models of ALI/ARDS are needed. Here we describe a semi-quantitative scoring system to evaluate histological injury using a previously established porcine model of ALI via intratracheal and intravascular lipopolysaccharide (LPS) administration. Additionally, and owing to the higher number of samples generated from large animal models, we worked to implement a more sustainable and greener histopathological workflow throughout the entire process.

Reduction of primary graft dysfunction using cytokine adsorption during organ preservation and after lung transplantation.

Despite improvements, lung transplantation remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine adsorption as a means of treating ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 16 donor pigs. Lungs were then treated with or without cytokine adsorption during ex vivo lung perfusion (EVLP) and/or post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine adsorption was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

Monitoring lung injury with particle flow rate in LPS- and COVID-19-induced ARDS.

In severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) is a life-prolonging treatment, especially among COVID-19 patients. Evaluation of lung injury progression is challenging with current techniques. Diagnostic imaging or invasive diagnostics are risky given the difficulties of intra-hospital transportation, contraindication of biopsies, and the potential for the spread of infections, such as in COVID-19 patients. We have recently shown that particle flow rate (PFR) from exhaled breath could be a noninvasive, early detection method for ARDS during mechanical ventilation. We hypothesized that PFR could also measure the progress of lung injury during ECMO treatment. Lipopolysaccharide (LPS) was thus used to induce ARDS in pigs under mechanical ventilation. Eight were connected to ECMO, whereas seven animals were not. In addition, six animals received sham treatment with saline. Four human patients with ECMO and ARDS were also monitored. In the pigs, as lung injury ensued, the PFR dramatically increased and a particular spike followed the establishment of ECMO in the LPS-treated animals. PFR remained elevated in all animals with no signs of lung recovery. In the human patients, in the two that recovered, PFR decreased. In the two whose lung function deteriorated while on ECMO, there was increased PFR with no sign of recovery in lung function. The present results indicate that real-time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS.

Increased particle flow rate from airways precedes clinical signs of ARDS in a porcine model of LPS-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is a common cause of death in the intensive care unit, with mortality rates of ~30-40%. To reduce invasive diagnostics such as bronchoalveolar lavage and time-consuming in-hospital transports for imaging diagnostics, we hypothesized that particle flow rate (PFR) pattern from the airways could be an early detection method and contribute to improving diagnostics and optimizing personalized therapies. Porcine models were ventilated mechanically. Lipopolysaccharide (LPS) was administered endotracheally and in the pulmonary artery to induce ARDS. PFR was measured using a customized particles in exhaled air (PExA 2.0) device. In contrast to control animals undergoing mechanical ventilation and receiving saline administration, animals who received LPS developed ARDS according to clinical guidelines and histologic assessment. Plasma levels of TNF-α and IL-6 increased significantly compared with baseline after 120 and 180 min, respectively. On the other hand, the PFR significantly increased and peaked 60 min after LPS administration, i.e., ~30 min before any ARDS stage was observed with other well-established outcome measurements such as hypoxemia, increased inspiratory pressure, and lower tidal volumes or plasma cytokine levels. The present results imply that PFR could be used to detect early biomarkers or as a clinical indicator for the onset of ARDS.

Ten year follow-up of lung transplantations using initially rejected donor lungs after reconditioning using ex vivo lung perfusion.

BACKGROUND: In 2006 and 2007 we performed double lung transplantation with marginal donor lungs assessed and reconditioned by Ex Vivo Lung Perfusion (EVLP), using a technique developed by Professor Stig Steen. Here we present a 10-year follow-up comparing the outcomes of lung transplantations performed at our clinic using EVLP lungs vs. conventional lungs. METHOD: Between 2006 and 2007, 21 patients (6 EVLP, 15 conventional) underwent double lung transplantation (LTx) with follow-up on May 2017 at Lund University Hospital, Sweden. Pulmonary function was measured at 3/6/12 months, and annually thereafter for a period of 10 years in addition to survival and freedom from chronic lung allograft dysfunction (CLAD) being analyzed. RESULTS: Regarding Forced Expiratory Volume in 1 s (FEV1) and 6MWT at 3, 6, and 12 months and annually thereafter, no difference in median FEV1 nor 6MWT was found for EVLP-LTx vs. conventional-LTx (p > 0.05). No difference was shown in post-operative survival between EVLP-LTx vs. conventional LTx for patients with an overall survival up to 10-years (p > 0.05). The same pattern was shown in sub analyses for patients with a limited survival up to 1 and 5 years (p > 0.05). CONCLUSION: No superiority was found in conventional-LTx over EVLP-LTx, neither in long-term survival nor pulmonary function. No difference in CLAD-free survival was seen between the two groups. We believe that EVLP is a safe and effective method to use in LTx, greatly increasing the donor pool by improving marginal lungs and providing an objective assessment of the viability of marginal donor lungs.

Different particle flow patterns from the airways after recruitment manoeuvres using volume-controlled or pressure-controlled ventilation.

OBJECTIVES: Noninvasive online monitoring of different particle flows from the airways may serve as an additional tool to assess mechanical ventilation. In the present study, we used a customised PExA, an optical particle counter for monitoring particle flow and size distribution in exhaled air, to analyse airway particle flow for three subsequent days. We compared volume-controlled ventilation (VCV) and pressure-controlled ventilation (PCV) and performed recruitment manoeuvres (RM). METHODS: Six animals were randomised into two groups: half received VCV before PCV and the other half received PCV before VCV. Measurements were taken daily for 1 h in each mode during three subsequent days in six fully anaesthetised domestic pigs. A RM was performed twice daily for 60 s at positive end-expiratory pressure (PEEP) of 10, 4 breaths/min and inspiratory-expiratory ratio (I:E) of 2:1. Measurements were taken for 3 min before the RM, 1 min during the RM and for 3 min after the RM. The particle sizes measured were between 0.48 and 3.37 µm. RESULTS: A significant stepwise decrease was observed in total particle count from day 1 to day 3, and at the same time, an increase in fluid levels was seen. Comparing VCV to PCV, a significant increase in total particle count was observed on day 2, with the highest particle count occurring during VCV. A significant increase was observed comparing before and after RM on day 1 and 2 but not on day 3. One animal developed ARDS and showed a different particle pattern compared to the other animals. CONCLUSIONS: This study shows the safety and useability of the PExA technique used in conjunction with mechanical ventilation. We detected differences between the ventilation modes VCV and PCV in total particle count without any significant changes in ventilator pressure levels, FiO2 levels or the animals' vital parameters. The findings during RM indicate an opening of the small airways, but the effect is short lived. We have also showed that VCV and PCV may affect the lung physiology differently during recruitment manoeuvres. These findings might indicate that this technique may provide more refined information on the impact of mechanical ventilation.

A new way of monitoring mechanical ventilation by measurement of particle flow from the airways using Pexa method in vivo and during ex vivo lung perfusion in DCD lung transplantation.

BACKGROUND: Different mechanical ventilation settings are known to affect lung preservation for lung transplantation. Measurement of particle flow in exhaled air may allow online assessment of the impact of ventilation before changes in the tissue can be observed. We hypothesized that by analyzing the particle flow, we could understand the impact of different ventilation parameters. METHODS: Particle flow was monitored in vivo, post mortem, and in ex vivo lung perfusion (EVLP) in six porcines with the Pexa (particles in exhaled air) instrument. Volume-controlled ventilation (VCV) and pressure-controlled ventilation (PCV) were used to compare small versus large tidal volumes. The surfactant lipids dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC) were quantified by mass spectrometry. RESULTS: In vivo the particle mass in VCV1 was significantly lower than in VCV2 (p = 0.0186), and the particle mass was significantly higher in PCV1 than in VCV1 (p = 0.0322). In EVLP, the particle mass in VCV1 was significantly higher than in PCV1 (p = 0.0371), and the particle mass was significantly higher in PCV2 than in PCV1 (p = 0.0127). DPPC was significantly higher in EVLP than in vivo. CONCLUSIONS: Here, we introduce a new method for measuring particle flow during mechanical ventilation and confirm that these particles can be collected and analyzed. VCV resulted in a lower particle flow in vivo but not in EVLP. In all settings, large tidal volumes resulted in increased particle flow. We found that DPPC was significantly increased comparing in vivo with EVLP. This technology may be useful for developing strategies to preserve the lung and has a high potential to detect biomarkers.

The impact of alteplase on pulmonary graft function in donation after circulatory death - An experimental study.

OBJECTIVE: Lung transplantation is hampered by the lack of organs resulting in deaths on the waiting list. The usage of donation after circulatory death (DCD) lungs would dramatically increase donor availability. The most optimal organ preservation method, and the need for antithrombotic and fibrinolytic treatment to prevent thrombosis in the donor lungs is currently on debate. The present study investigated, in a simulated clinical DCD situation, whether the addition of alteplase in the flush-perfusion solution at the time of pulmonary graft harvesting could prevent thrombosis in the donor lung and thereby improve pulmonary graft function. METHODS: Twelve Swedish domestic pigs were randomized into two groups. All animals underwent ventricular fibrillation and were then left untouched for 1 h after declaration of death. None of the animals received heparin. The lungs were then harvested and flush-perfused with Perfadex® solution and the organs were then stored at 8 °C for 4 h. In one group alteplase was added to the Perfadex® solution (donation after cardiac death with alteplase (DCD-A)) and in the other, it was not (DCD). Lung function was evaluated, using ex vivo lung perfusion (EVLP), with blood gases at different oxygen levels, pulmonary vascular resistance (PVR), lung weight, and macroscopic appearance. RESULTS: During EVLP, there were no significant differences between groups in PaO2 at any investigated FiO2 level (1.0, 0.5, or 0.21). At FiO2 1.0, the PaO2 in the DCD and DCD-A was 51.7 ± 2.05 kPa and 60.3 ± 3.67 kPa, respectively (p = 0.1320). There were no significant differences between groups PVR levels, in the DCD (372 ± 31 dyne x s/cm5) and in the DCD-A (297 ± 37 dyne x s/cm5) groups (p = 0.1720). There was no significant difference between groups in macroscopic appearance. CONCLUSIONS: All the lungs showed excellent blood gases after EVLP, and they all meet the criteria's for clinical lung transplantation. The use of alteplase did not seem to have any obvious benefit to the donor lungs in a DCD situation. The donor lungs treated with alteplas showed slightly better blood gases and slightly lower PVR compared to the group without alteplas, however the difference was not significant. DCD appears to be a safe and effective method to expand the donor pool.

Protection of pulmonary graft from thrombosis in donation after cardiac death: effect of warm ischaemia versus cold ischaemia.

OBJECTIVES: The use of donation after cardiac death (DCD) to overcome organ shortage is slowly moving into the clinic. In this study, we compare the protective effect of warm ischaemia versus cold ischaemia on thrombotic formation in non-heparinized pulmonary grafts. METHODS: Twelve Landrace pigs were randomized into two groups: warm ischaemia and cold ischaemia. Ventricular fibrillation without the administration of heparin was induced to mimick an uncontrolled DCD situation. The animals were then exposed to either 1 h of cold ischaemia (insertion of drain and installation of cold fluid in the pleuras) or warm ischaemia (body temperature). After 1 h, the pulmonary artery was opened and the pulmonary arterial branches were then macroscopically studied for thrombotic material. RESULTS: After 60 min, the temperature was 36.6 ± 0.0°C in the warm ischaemic group and 14.6 ± 0.1°C in the cold ischaemic group (P < 0.001). In the warm ischaemic group, no thrombotic material could be found in the pulmonary artery in the animals examined and in the cold ischaemic group 6.8 ± 0.2 ml thrombotic material was found in the pulmonary artery (P < 0.001). In the warm ischaemic group, no thrombotic material could be found in the arterial branches of the pulmonary artery and in the cold ischaemic group 2.3 ± 0.1 ml thrombotic material was found in the arterial branches of the pulmonary artery (P < 0.001). CONCLUSIONS: Warm ischaemia rather than cold ischaemia seems to protect the pulmonary graft from thrombosis in uncontrolled non-heparinized DCD pigs.

Pulmonary collapse alone provides effective de-airing in cardiac surgery: a prospective randomized study.

OBJECTIVES: We previously described and showed that the method for cardiac de-airing involving: (1) bilateral, induced pulmonary collapse by opening both pleurae and disconnecting the ventilator before cardioplegic arrest and (2) gradual pulmonary perfusion and ventilation after cardioplegic arrest is superior to conventional de-airing methods, including carbon dioxide insufflation of the open mediastinum. This study investigated whether one or both components of this method are responsible for the effective de-airing of the heart. METHODS: Twenty patients scheduled for open, left heart surgery were randomized to two de-airing techniques: (1) open pleurae, collapsed lungs and conventional pulmonary perfusion and ventilation; and (2) intact pleurae, expanded lungs and gradual pulmonary perfusion and ventilation. RESULTS: The number of cerebral microemboli measured by transcranial Doppler sonography was lower in patients with open pleurae 9 (6-36) vs 65 (36-210), p = 0.004. Residual intra-cardiac air grade I or higher as monitored by transesophageal echocardiography 4-6 minutes after weaning from cardiopulmonary bypass was seen in few patients with open pleurae 0 (0%) vs 7 (70%), p = 0.002. CONCLUSIONS: Bilateral, induced pulmonary collapse alone is the key factor for quick and effective de-airing of the heart. Gradual pulmonary perfusion and ventilation, on the other hand, appears to be less important.

Lungs exposed to 1 hour warm ischemia without heparin before harvesting might be suitable candidates for transplantation.

BACKGROUND: The limiting factor for lung transplantation is the lack of donor organs. The usage of lungs from donation after cardiac death (DCD) would dramatically increase donor availability. In the present paper we wanted to investigate lungs exposed to 1 h of warm ischemia without heparin followed by flush-perfusion and cold storage compared to lungs harvested from heart beating donors (HBD) using standard harvesting technique. METHODS: Twelve Swedish domestic pigs were randomized into two groups. Six pigs (DCD group) underwent ventricular fibrillation and were then left untouched for 1 h after declaration of death. They did not receive heparin. The lungs were then harvested and flush-perfused with Perfadex® solution and the organs were stored at 8 °C for 4 h. Six pigs (HBD group) received heparin and the lungs were harvested and flush-perfused with Perfadex® solution and the organs were stored at 8 °C for 4 h. Lung function was evaluated, using ex vivo lung perfusion (EVLP), with blood gases at different oxygen levels, pulmonary vascular resistance (PVR), lung weight, and macroscopic appearance. RESULTS: At FiO2 1.0, the PaO2 in the DCD group was 51.7 ± 2.0 kPa and in the HBD group 68.6 ± 2.4 kPa (p < 0.01). Significantly lower PVR levels were measured in the DCD group (372 ± 31 dyne x s/cm(5)) compared to the HBD group (655 ± 45 dyne x s/cm(5)) (p < 0.001). There was no significant difference between groups in weight, compliance or signs of pulmonary thrombosis or embolization. CONCLUSIONS: It seems as if DCD lungs exposed to 1 h of warm ischemia before 4 h of cold storage has satisfying oxygenation capacity, low PVR, normal weight and no signs of thrombosis or embolization. According to our study it seems as lungs exposed to 1 h warm ischemia without heparin might be good candidates for transplantation.

Ventilation in situ after cardiac death improves pulmonary grafts exposed to 2 hours of warm ischemia.

BACKGROUND: The pulmonary donor pool would increase substantially if lungs could be donated after cardiac death (DCD). There have been ethical and legal obstacles since administration of heparin and cooling has to be done immediately after cardiac death. This study examines whether ventilation of DCD lungs without administering heparin or cooling the lungs after cardiac death could improve graft function. METHOD: Twelve donor pigs with a mean bodyweight of 70 kg were randomized into two groups. Six animals were ventilated in situ with 50% oxygen, 4 L/min, and 5 cm H2O in positive end-expiratory pressure or PEEP for 2 h after cardiac death. Six animals served as non-ventilated controls and were exposed to warm ischemia for 2 h. After 2 h, all lungs were harvested and flush perfused with Perfadex(®) solution and stored at 8°C for another 2 h. An ex vivo lung perfusion or EVLP circuit was used for evaluation. RESULTS: Non-ventilated lungs developed pulmonary edema, and had highly impaired blood gas levels and a significantly increased weight. The ventilated lungs demonstrated excellent blood gas levels and unchanged weight. CONCLUSION: The increase in tolerable warm ischemic time in combination with avoiding heparinization and cooling might facilitate the use of DCD lungs for transplantation.

Systemic effects of carbon dioxide insufflation technique for de-airing in left-sided cardiac surgery.

OBJECTIVE: Systemic effects of carbon dioxide (CO2) insufflation during left-sided cardiac surgery were evaluated in a prospective randomized study, with regard to acid-base status, gas exchange, cerebral hemodynamics, and red blood cell morphology. METHODS: Twenty patients undergoing elective left-sided cardiac surgery were randomized to de-airing procedure either by CO2 insufflation technique (CO2 group, n = 10) or by Lund technique without CO2 insufflation (Lund group, n = 10). Groups underwent assessment of acid-base status by intermittent arterial blood gases and in-line blood gas monitoring. Capnography was used to determine volume of CO2 produced. Cerebral hemodynamics was measured by transcranial Doppler sonography and near-infrared spectroscopy. Red cell morphology from cardiotomy suction and vent tubing was studied by scanning electron microscopy. RESULTS: Patients in the CO2 group consequently developed significantly higher levels of hypercapnia with a concomitant increase in the volume of CO2 produced despite significantly higher oxygenator gas flows compared with the Lund group. Effects on cerebral hemodynamics were observed in the CO2 group with significantly higher blood flow velocities in the middle cerebral artery and higher regional cerebral saturation. Red blood cell damage was observed in the CO2 group by scanning electron microscopy (97% in CO2 group vs 18% in Lund group). CONCLUSIONS: Insufflation of CO2 into the cardiothoracic wound cavity during left-sided cardiac surgery can induce hypercapnic acidosis and increased cerebral blood flow and local blood cell damage. These systemic effects should be monitored by in-line capnography and acid-base measurements for early and effective correction by increase in gas flows to the oxygenator.