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Ex vivo lung perfusion (EVLP) is an emerging procedure that allows organ preservation, assessment and reconditioning, increasing the number of marginal donor lungs for transplantation. However, physiological and airflow measurements are unable to unveil the molecular mechanisms responsible of EVLP beneficial effects on lung graft and monitor the proper course of the treatment. Thus, it is urgent to find specific biomarkers that possess these requirements but also accurate and reliable techniques that identify them. The purpose of this study is to give an overview on the potentiality of shotgun proteomic platforms in characterizing the status and the evolution of metabolic pathways during EVLP in order to find new potential EVLP-related biomarkers. A nanoLC-MS/MS system was applied to the proteome analysis of lung tissues from an optimized rat model in three experimental groups: native, pre- and post-EVLP. Technical and biological repeatability were evaluated and, together with clustering analysis, underlined the good quality of data produced. In-house software and bioinformatics tools allowed the label-free extraction of differentially expressed proteins among the three examined conditions and the network visualization of the pathways mainly involved. These promising findings encourage further proteomic investigations of the molecular mechanisms behind EVLP procedure.

Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

Ex vivo lung perfusion (EVLP) is a promising procedure for evaluation, reconditioning, and treatment of marginal lungs before transplantation. Small animal models can contribute to improve clinical development of this technique and represent a substantial platform for bio-molecular investigations. However, to accomplish this purpose, EVLP models must sustain a prolonged reperfusion without pharmacological interventions. Currently available protocols only partly satisfy this need. The aim of the present research was accomplishment and optimization of a reproducible model for a protracted rat EVLP in the absence of anti-inflammatory treatment. A 180 min, uninjured and untreated perfusion was achieved through a stepwise implementation of the protocol. Flow rate, temperature, and tidal volume were gradually increased during the initial reperfusion phase to reduce hemodynamic and oxidative stress. Low flow rate combined with open atrium and protective ventilation strategy were applied to prevent lung damage. The videos enclosed show management of the most critical technical steps. The stability and reproducibility of the present procedure were confirmed by lung function evaluation and edema assessment. The meticulous description of the protocol provided in this paper can enable other laboratories to reproduce it effortlessly, supporting research in the EVLP field., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Uncontrolled donors after circulatory determination of death (uDCDD) represent a yet unexplored pool of organs potentially available for transplantation. The aims of this study were to validate a protocol of cardiac death in the pig and to investigate lung function during the process., Materials and Methods: Cardiac death was induced in preanesthetized animals with an injection of 600 mg propofol; once systolic blood pressure was <50 mm Hg (Agonal Phase), a 20 mEq bolus of KCl was given and, after asystolia was documented, cardiopulmonary resuscitation (CPR) started, followed by 5 minutes no touch (end-CPR). Invasive blood pressure (BP) and heart rate (HR) were recorded; blood samples taken at baseline, 15 minutes after CPR, and after the no touch period (end-CPR). Computed tomography (CT) scans were taken at baseline and at end-CPR., Results: Agonal phase was reached in 6 ± 1 minutes and lasted 3 ± 1 minutes; average HR was 49 ± 16 beats/min, and BP was 41 ± 12 mm Hg. CPR lasted 35 ± 3 minutes; average HR and BP were 113 ± 32 beats/min and 86 ± 63 mm Hg, respectively. PaO2/FiO2 decreased from 442 ± 31 mm Hg at baseline to 63 ± 36 at end-CPR (P < .001). pH decreased from 7.378 ± 0.045 to 6.931 ± 0.042 (P < .001), with a corresponding increase of lactate from 0.9 ± 0.2 to mmol/L to 12.8 ± 2.1 (P < .001). As assessed using CT scan, total lung volume decreased (baseline vs end-CPR 1107 ± 106 mL vs 617 ± 95; P < .001), whereas noninflated tissue (ie, atelectasis) significantly increased (46 ± 10 g vs 131 ± 89; P = .008)., Conclusions: Lung function greatly deteriorated after cardiac death. The model we set may constitute a reproducible platform for future investigations on lung uDCDD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Among patients with respiratory insufficiency awaiting lung transplantation, small adult patients have a lower opportunity of receiving size-matched pulmonary grafts, because of the shortage of donors, particularly those of small size. Reducing the size of an oversized graft is one of the methods to increase the donor pool; similarly, ex vivo lung perfusion is an emerging technique aimed toward the same purpose. We describe how we combined the 2 techniques (lobar transplantation plus contralateral nonanatomic graft reduction during ex vivo lung perfusion) to overcome graft shortage in a clinical case. For the 1st time, this case report demonstrates that surgical manipulation during ex vivo lung perfusion does not affect the functional improvement in a lung previously judged to be not suitable for transplantation. The 6-month follow-up results are similar to those of standard bilateral lung transplantation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Ex vivo lung perfusion (EVLP) has been validated as a valuable technique to increase the pool of organs available for lung transplantation., Material and Methods: After a preclinical experience, we obtained permission from the Ethics Committee of our institution to transplant lungs after EVLP reconditioning. ABO compatibility, size match, and donor arterial oxygen pressure (PaO(2))/fraction of inspired oxygen (FiO(2)) ≤ 300 mm Hg were considered to be inclusion criteria, whereas the presence of chest trauma and lung contusion, evidence of gastric content aspiration, pneumonia, sepsis, or systemic disease were exclusion criteria. We only considered subjects on an extra corporeal membrane oxygenation (ECMO) bridge to transplantation with rapid functional deterioration. Using Steen solution with packed red blood cells oxygenated with 21% O(2), 5% to 7% CO(2) was delivered, targeted with a blood flow of approximately 40% predicted cardiac output. Once normothermic, the lungs were ventilated with a tidal volume of 7 mL/kg a PEEP of 5 cmH(2)O and a respiratory rate of 7 bpm. Lungs were considered to be suitable for transplantation if well oxygenated [P(v-a) O(2) > 350 mm Hg on FiO(2) 100%], in the absence of deterioration of pulmonary vascular resistance and lung mechanics over the perfusion time., Results: From March to September 2011, six lung transplantations were performed, including two with EVLP. The functional outcomes were similar between groups: at T72 posttransplantation, the median PaO(2)/FiO(2) were 306 mm Hg (range, 282 to 331 mm Hg) and 323 mm Hg (range, 270 to 396 mm Hg) (P = 1, EVLP versus conventional). Intensive care unit ICU and hospital length of stay were similar (P = .533 and P = .663, respectively) with no mortality at 60 days in both groups. EVLP donors were older (49 ± 6 y versus 21 ± 7 y, P < .05), less well oxygenated (184 ± 6 mm Hg versus 570 ± 30, P < .05), displaying higher Oto scores (9.5 ± 0.7 versus 1.7 ± 1.5, P < .05)., Conclusions: The first 6 months of the EVLP program allowed us to increase the number of organs available for transplantation with short-term outcomes comparable to conventional transplantations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: We recently showed in a pig model of ex vivo lung perfusion (EVLP) that lung edema correlates with glucose consumption. We investigated whether salbutamol, a β-adrenergic receptor agonist known to upregulate fluid transport in the lung, modulates glucose concentration in the perfusate during EVLP., Methods: Lungs from domestic pigs underwent normothermic EVLP. At the end of controlled reperfusion, lungs were ventilated and perfused for 60 minutes, then randomized to salbutamol (β-Agonist) infusion or placebo (Control) for 180 minutes. Functional parameters were assessed., Results: In the β-Agonist group, glucose concentration decreased over time more than corresponding Control values (analysis of variance [ANOVA], p = 0.05). Mean pulmonary artery pressure (mPAP) was 16 ± 1 mm Hg in the β-Agonist group vs 21 ± 1 mm Hg in the Controls (ANOVA p < 0.05). Baseline mPAP was correlated with the drop of mPAP after the β-agonist infusion (R(2) = 0.856, p < 0.05). Dynamic compliance dropped from 51 ± 10 to 31 ± 6 ml/cm H(2)O in the β-Agonist group and from 60 ± 4 to 21 ± 3 ml/cm H(2)O in the Control group (ANOVA, p < 0.05 β-agonist vs Control). The Δ partial pressure of oxygen/fraction of inspired oxygen was 418 ± 15 and 393 ± 12 mm Hg in the β-Agonist and Control groups, respectively (t-test p = 0.106)., Conclusions: Glucose concentration in the perfusate was affected by salbutamol. Salbutamol was associated with lower pulmonary pressures and better lung mechanics. These data suggest a possible role for salbutamol as a pharmacologic adjunct during EVLP before transplantation., (Copyright © 2012 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Introduction: Ex vivo lung perfusion (EVLP) has been recently proposed to recondition organs before transplantation from donors with marginal or unacceptable features. The aim of our investigation was to explore glucose consumption during EVLP., Materials and Methods: We investigated 8 domestic pigs (mean weight, 21 ± 0.8 kg). After perfusion with Perfadex, retrieval, and back table surgery, we initiated EVLP. The lungs were perfused with Steen solution with added methylprednisolone, cefazoline, and heparin. The blood flow was gradually increased with a target of 40% of the estimated cardiac output (or less if the pulmonary artery pressure was >15 mm Hg), while keeping the left atrial pressure between 3 and 5 mm Hg. The temperature of the perfusate was increased from 25 °C to 37 °C. Once the temperature of the lung outflow was >32 °C, we began gas flow (4 L/min, 5%-8% CO(2) in air) and mechanical ventilation. EVLP parameters and blood gases were measured throughout the experiment; glucose consumption was calculated as (glucose initial-glucose final)/time. The wet to dry ratio was also calculated as an index of lung edema., Results: When stratified by median glucose consumption (0.237 mg/min), high glucose consumers (0.588 ± 0.17) were characterized by worse lung function, as assessed by oxygenation (partial pressure of oxygen/inspiratory fraction of oxygen [PaO(2)/FiO(2)] 326 ± 63 mm Hg vs 218 ± 84; P=.083 low vs high, respectively), and lung edema (wet/dry ratio 6.5 ± 0.7 vs 8.6 ± 0.9; P=.012). Glucose consumption correlated with wet to dry ratio (R(2)=0.663; P=.014)., Conclusions: We found that the worse the lung function, the greater the consumption of glucose during EVLP. This observation suggests the need to explore lung metabolism during EVLP to possibly obtain metrics for evaluation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To investigate if prone position delays the progression of experimental ventilator-induced lung injury, possibly due to a more homogeneous distribution of strain within lung parenchyma., Design: Prospective, randomized, controlled trial., Setting: Animal laboratory of a university hospital., Subjects: Thirty-five Sprague Dawley male rats (weight 257 +/- 45 g)., Interventions: Mechanical ventilation in either supine or prone position and computed tomography scan analysis., Measurements: : Animals were ventilated in supine (n = 15) or prone (n = 15) position until a similar ventilator-induced lung injury was reached. To do so, experiments were interrupted when respiratory system elastance was 150% of baseline. Ventilator-induced lung injury was assessed as lung wet-to-dry ratio and histology. Time to reach lung injury was considered as a main outcome measure. In five additional animals, computed tomography scans (GE Light Speed QX/I, thickness 1.25 mm, interval 0.6 mm, 100 MA, 100 Kv) were randomly taken at end-expiration and end-inspiration in both positions, and quantitative analysis was performed. Data are shown as mean +/- sd., Measurements and Main Results: Similar ventilator-induced lung injury was reached (respiratory system elastance, wet-to-dry ratio, and histology). The time taken to achieve the target ventilator-induced lung injury was longer with prone position (73 +/- 37 mins vs. 112 +/- 42, supine vs. prone, p = .011). Computed tomography scan analysis performed before lung injury revealed that at end-expiration, the lung was wider in prone position (p = .004) and somewhat shorter (p = .09), despite similar lung volumes (p = .455). Lung density along the vertical axis increased significantly only in supine position (p = .002). Lung strain was greater in supine as opposed to prone position (width strain, 7.8 +/- 1.8% vs. 5.6 +/- 0.9, supine vs. prone, p = .029)., Conclusions: Prone position delays the progression of ventilator-induced lung injury. Computed tomography scan analysis suggests that a more homogeneous distribution of strain may be implicated in the protective role of prone position against ventilator-induced lung injury.

In the last several years, we definitely learned that the acute respiratory distress syndrome lung is small, nonhomogeneous, and that mechanical ventilation in this baby lung may cause physical damage as well as inflammatory reaction. The clinical benefit of the gentle lung treatment, based on a decrease of global/regional stress and strain into the lung, has been finally proved. However, we forgot the importance of lung perfusion and its distribution in this syndrome and, besides a low tidal volume, we still do not know how to handle the other variables of mechanical ventilation. Measurements of variables as transpulmonary pressure and end expiratory lung volume, for a rational setting of mechanical ventilation, should be introduced in routine clinical practice.

We examined the effect of ventilation strategy on lung inflammatory mediators in the presence and absence of a preexisting inflammatory stimulus. 55 Sprague-Dawley rats were randomized to either intravenous saline or lipopolysaccharide (LPS). After 50 min of spontaneous respiration, the lungs were excised and randomized to 2 h of ventilation with one of four strategies: (a) control (C), tidal volume (Vt) = 7 cc/kg, positive end expiratory pressure (PEEP) = 3 cm H2O; (b) moderate volume, high PEEP (MVHP), Vt = 15 cc/kg; PEEP = 10 cm H2O; (c) moderate volume, zero PEEP (MVZP), Vt = 15 cc/kg, PEEP = 0; or (d) high volume, zero PEEP (HVZP), Vt = 40 cc/kg, PEEP = 0. Ventilation with zero PEEP (MVZP, HVZP) resulted in significant reductions in lung compliance. Lung lavage levels of TNFalpha, IL-1beta, IL-6, IL-10, MIP-2, and IFNgamma were measured by ELISA. Zero PEEP in combination with high volume ventilation (HVZP) had a synergistic effect on cytokine levels (e.g., 56-fold increase of TNFalpha versus controls). Identical end inspiratory lung distention with PEEP (MVHP) resulted in only a three-fold increase in TNFalpha, whereas MVZP produced a six-fold increase in lavage TNFalpha. Northern blot analysis revealed a similar pattern (C, MVHP < MVZP < HVZP) for induction of c-fos mRNA. These data support the concept that mechanical ventilation can have a significant influence on the inflammatory/anti-inflammatory milieu of the lung, and thus may play a role in initiating or propagating a local, and possibly systemic inflammatory response.

The distribution of tidal volume (VT) and recruitment was investigated by chest computed tomography (CT) in eight sedated-paralyzed patients with the adult respiratory distress syndrome (ARDS). A CT section was obtained in the supine position at 0, 5, 10, 15, and 20 cm H2O positive end-expiratory pressure (PEEP) and at the corresponding inspiratory plateau pressure (21 +/- 1.8, 26 +/- 1.4, 31 +/- 1.8, 38 +/- 2.1, and 46 +/- 3.2 cm H2O [mean +/- SE]), keeping VT constant. Each CT section was divided along its ventral-dorsal height into 10 equally spaced intervals (levels). Vi(insp) and Vi(exp) were defined as the gas volume for level i (i = 1 to 10) at end-inspiration and at end-expiration, respectively. The following variables were computed at each lung level: (1) distribution of CT section tidal volume (VTct), i.e., the fraction of VT that inflates a given lung level; (2) the plateau-induced and PEEP-induced recruitment, i.e., the amount of lung tissue previously collapsed that inflates at plateau pressure and at PEEP, respectively; (3) the reopening-collapsing tissue, i.e., the amount of lung tissue that regains inflation at plateau pressure and collapses at PEEP. With increasing PEEP from 0 to 20 cm H2O, the VTct distribution decreased significantly (p < 0.01) in the upper levels, did not change in the middle levels, and increased significantly (p < 0.01) in the lower levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric Oxide (NO) has been implicated in the pathologic vasodilation of sepsis. Because NO can be measured in the exhaled gas of animals and humans, we hypothesized that increases in exhaled NO would occur in a septic model. Using a blinded design, 10 male Sprague-Dawley rats (300 to 400 g) were anesthetized, paralyzed, tracheotomized, and randomized (5/group) to receive an intravenous injection of either lipopolysaccharide (LPS) (Salmonella typhosa, 20 mg/kg) or placebo (equal volume of saline). Thereafter, exhaled gas was collected and measurements of NO concentration were made using chemiluminescence every 20 min for 300 min during ventilation (RR 40 breaths/min, VT 3 ml; PEEP 0, FIO2 0.21). Another group of 10 animals (5 LPS; 5 control) were treated in the same fashion and then killed at 240 min and an arterial blood sample obtained for blood gas and TNF alpha determinations. Pressure volume (PV) curves were constructed and lungs removed, preserved, and submitted for histologic evaluation. LPS-treated rats had lower mean arterial pressures than the control group, p < 0.0001. No significant differences in static lung compliance and PV curves were found in the two groups. TNF alpha levels were greater in the LPS group (1.40 +/- 0.24 ng/ml) versus control group (0.09 +/- 0.04 ng/ml), p < 0.001. By contrast to the control group, exhaled NO concentration rose in all LPS-treated rats at approximately 100 min and at about 160 min reached a plateau that was 6 times greater than control levels (p < 0.0001). There was greater interstitial, airspace, and total lung injury in the LPS group (p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)