Porcine Lung Research Articles

BIO24: Reducing Risk of Barotrauma in Donor Lungs During Transport

Background: The journey donor lungs take after being procured on the way to the recipient often involves the use of air transportation, subjecting the organ to different altitudes. During takeoff and at cruising altitudes, decreased atmospheric pressures lead to increased relative airway pressures, increasing the risk for barotrauma. While this may be due to underinflation during procurement, the risk of barotrauma likely still exists given the subjective nature of the process and variability between surgeons and centers. Methods: Using porcine lungs (n=3), the airway pressure of donor lungs in simulated flight takeoff and landing was actively controlled using a novel preservation system and measured with calibrated gauges. Lungs were prepared using 2L preservation solution at 4°C in the inner bag and 2L saline at 4°C in the middle bag. A modified 3 bag system allowed for the trachea to be connected to pneumatic circuit designed to maintain target airway pressures of 12-15 cmH2O per the ISHLT consensus statement. Parameters for simulated takeoff and landing (1,000ft/min), peak altitude (8,000ft), and time at peak altitude (6 hours) were chosen to reflect the worst-case scenario. Results: All airway pressures remained within 12-15 cmH2O (±1 cmH2O) following a maximum of 5-minute stabilization time after transitioning between stable ambient pressures. This demonstrates the ability of the system to maintain airway pressures within this range at pressures up to 8000ft (worst-case for pressurized aircraft cabin). During simulated takeoff and landing scenarios, all airway pressures remained within 10-20 cmH2O at 1000ft. Without active pressure control, airway pressures may increase above 30 cmH2O. Conclusions: Airway pressures during flight remained within the intended range using a novel preservation system for donor lungs. Without active control using the systems, airway pressure of lungs are subjected to unsafe levels, which may lead to suboptimal postoperative outcomes.

Compression Behavior of Extra-soft Porcine Lung Tissue at the Wide Strain Rate

Lung is the respiratory organ and the important hematopoietic organ of the human body, which is easily damaged under impact load. Therefore, studying the mechanical behavior of the lung under impact load is an important way to understand its damage mechanism of it. Compression experiments of porcine lung tissue at a wide strain rate (0.001-3000 s−1) were carried out at room temperature. The modifications were made for the highly accurate mechanical measurement of the tissue due to its low wave impedance. The results show that its behaviors consist of three stages, linear elasticity, plateau stage, and compaction. The lung tissue presents distinct rate-dependence in the compaction stage. Furthermore, it behaves nearly similarly under both the loading directions of vertical and parallel trachea, which facilitates to a demonstration of its isotropy.

In vivo solid phase microextraction for therapeutic monitoring and pharmacometabolomic fingerprinting of lung during in vivo lung perfusion of FOLFOX

In vivo lung perfusion (IVLP) is a novel isolated lung technique developed to enable the local, in situ administration of high-dose chemotherapy to treat metastatic lung cancer. Combination therapy using folinic acid (FOL), 5-fluorouracil (F), and oxaliplatin (OX) (FOLFOX) is routinely employed to treat several types of solid tumours in various tissues. However, F is characterized by large interpatient variability with respect to plasma concentration, which necessitates close monitoring during treatments using of this compound. Since plasma drug concentrations often do not reflect tissue drug concentrations, it is essential to utilize sample-preparation methods specifically suited to monitoring drug levels in target organs. In this work, in vivo solid-phase microextraction (in vivo SPME) is proposed as an effective tool for quantitative therapeutic drug monitoring of FOLFOX in porcine lungs during pre-clinical IVLP and intravenous (IV) trials. The concomitant extraction of other endogenous and exogenous small molecules from the lung and their detection via liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) enabled an assessment of FOLFOX's impact on the metabolomic profile of the lung and revealed the metabolic pathways associated with the route of administration (IVLP vs. IV) and the therapy itself. This study also shows that the immediate instrumental analysis of metabolomic samples is ideal, as long-term storage at −80 °C results in changes in the metabolite content in the sample extracts.

(235) Neutrophil Extracellular Traps Removal During Ex Vivo Lung Perfusion Improves Lung Function in Aspiration Damaged Porcine Lungs

(235) Neutrophil Extracellular Traps Removal During Ex Vivo Lung Perfusion Improves Lung Function in Aspiration Damaged Porcine Lungs

A feasible technique for distinguishing the intersegmental plane by transbronchial injection of iron sucrose

Pulmonary segmentectomy should be the standard surgical procedure for patients in certain clinical scenarios. However, detecting the intersegmental planes both on the pleural surface and within the lung parenchyma remains a challenge. We developed an intraoperative novel method for distinguishing intersegmental planes of the lung via transbronchial injection of iron sucrose (ClinicalTrials.gov number, NCT03516500). We first performed a bronchial injection of iron sucrose to identify the intersegmental plane of the porcine lung. Then, we conducted a prospective study to evaluate the safety and feasibility of the technique in 20 patients who underwent anatomic segmentectomy. Iron sucrose was injected into the bronchus of target pulmonary segments, and the intersegmental planes were divided with electrocautery or stapler. The median injection of iron sucrose was 90mL (range, 70-120mL), and the median time from injection of iron sucrose to demarcation of intersegmental plane was 8minutes (range, 3-25minutes). Qualified identification of the intersegmental plane was observed in 17 cases (85%). The intersegmental plane could not be recognized in 3 cases. All patients experienced no complications related to iron sucrose injection or complications of Clavien-Dindo grade 3 or more. Transbronchial injection of iron sucrose is a simple, safe, and feasible approach to identify the intersegmental plane (NCT03516500).

High resolution propagation-based lung imaging at clinically relevant X-ray dose levels

Absorption-based clinical computed tomography (CT) is the current imaging method of choice in the diagnosis of lung diseases. Many pulmonary diseases are affecting microscopic structures of the lung, such as terminal bronchi, alveolar spaces, sublobular blood vessels or the pulmonary interstitial tissue. As spatial resolution in CT is limited by the clinically acceptable applied X-ray dose, a comprehensive diagnosis of conditions such as interstitial lung disease, idiopathic pulmonary fibrosis or the characterization of small pulmonary nodules is limited and may require additional validation by invasive lung biopsies. Propagation-based imaging (PBI) is a phase sensitive X-ray imaging technique capable of reaching high spatial resolutions at relatively low applied radiation dose levels. In this publication, we present technical refinements of PBI for the characterization of different artificial lung pathologies, mimicking clinically relevant patterns in ventilated fresh porcine lungs in a human-scale chest phantom. The combination of a very large propagation distance of 10.7 m and a photon counting detector with 100,upmu hbox {m} pixel size enabled high resolution PBI CT with significantly improved dose efficiency, measured by thermoluminescence detectors. Image quality was directly compared with state-of-the-art clinical CT. PBI with increased propagation distance was found to provide improved image quality at the same or even lower X-ray dose levels than clinical CT. By combining PBI with iodine k-edge subtraction imaging we further demonstrate that, the high quality of the calculated iodine concentration maps might be a potential tool for the analysis of lung perfusion in great detail. Our results indicate PBI to be of great value for accurate diagnosis of lung disease in patients as it allows to depict pathological lesions non-invasively at high resolution in 3D. This will especially benefit patients at high risk of complications from invasive lung biopsies such as in the setting of suspected idiopathic pulmonary fibrosis (IPF).

Real-time Lung Weight Measurement During Cellular Ex Vivo Lung Perfusion: An Early Predictor of Transplant Suitability.

Increased extravascular lung water during ex vivo lung perfusion (EVLP) is associated with ischemia reperfusion injury and poor pulmonary function. A non-invasive technique for evaluating extravascular lung water during EVLP is desired to assess the transplant suitability of lungs. We investigated real-time lung weight measurements as a reliable method for assessing pulmonary functions in cellular EVLP using a porcine lung model. Fifteen pigs were randomly divided into 3 groups: control (no warm ischemia) or donation after circulatory death groups with 60 or 90 min of warm ischemia (n = 5, each). Real-time lung weight gain was measured by load cells positioned at the bottom of the organ chamber. Real-time lung weight gain at 2 h was significantly correlated with lung weight gain as measured on a back table ( R = 0.979, P < 0.01). Lung weight gain in non-suitable cases (n = 6) was significantly higher than in suitable cases (n = 9) at 40 min (51.6 ± 46.0 versus -8.8 ± 25.7 g; P < 0.01, cutoff = +12 g, area under the curve = 0.907). Lung weight gain at 40 min was significantly correlated with PaO 2 /FiO 2 , peak inspiratory pressure, shunt ratio, wet/dry ratio, and transplant suitability at 2 h ( P < 0.05, each). In non-suitable cases, lung weight gain at 66% and 100% of cardiac output was significantly higher than at 33% ( P < 0.05). Real-time lung weight measurement could potentially be an early predictor of pulmonary function in cellular EVLP.

The choice of an autocorrelation length in dark-field lung imaging

Respiratory diseases are one of the most common causes of death, and their early detection is crucial for prompt treatment. X-ray dark-field radiography (XDFR) is a promising tool to image objects with unresolved micro-structures such as lungs. Using Talbot-Lau XDFR, we imaged inflated porcine lungs together with Polymethylmethacrylat (PMMA) microspheres (in air) of diameter sizes between 20 and 500 upmu hbox {m} over an autocorrelation range of 0.8–5.2 upmu hbox {m}. The results indicate that the dark-field extinction coefficient of porcine lungs is similar to that of densely-packed PMMA spheres with diameter of {200},upmu hbox {m}, which is approximately the mean alveolar structure size. We evaluated that, in our case, the autocorrelation length would have to be limited to {0.57},upmu hbox {m} in order to image {20},hbox {cm}-thick lung tissue without critical visibility reduction (signal saturation). We identify the autocorrelation length to be the critical parameter of an interferometer that allows to avoid signal saturation in clinical lung dark-field imaging.

Stability and safety of transbronchial dye mixture for preoperative localization in a porcine model.

For thoracoscopy, the usefulness of a dye mixture of indigo carmine and Lipiodol for localizing lung lesions has been reported. However, little is known about the stability and safety of this dye mixture injected on the visceral pleura through a bronchoscope. Porcine models were divided into three groups according to the detection time of the dye mixture: group A with a detection time of 4h; group B, 8h; and group C, 24 h. A dye mixture of indigo carmine and Lipiodol (0.5mL each) was sprayed onto the visceral pleura both in the ventral and dorsal regions via a spray catheter. Twelve markings were created on the visceral pleura of the porcine lung (six ventral and six dorsal) in the six porcine models. At predetermined detection times, all 12 dye markings (100%) were visible on the visceral pleura. The mean longest diameter of the dye marking in the ventral and dorsal regions was 18.8 mm and 24.3 mm, respectively. In groups B and C, pathological changes in the lymphatic system, such as lymphatic dilatations, were found; minimal changes were found in group B, however, these changes with oval-shaped lymphatic cysts and Lipiodol accumulation, were more evident in group C. The dye mixture of indigo carmine and Lipiodol had reliable stability and visibility. In terms of safety, it may be necessary to check the dye mixture on the lung surface within 8h.

L-alanyl-L-glutamine modified perfusate improves human lung cell functions and extend porcine ex vivo lung perfusion.

The clinical application of normothermic ex vivo lung perfusion (EVLP) has increased donor lung utilization for transplantation through functional assessment. To develop it as a platform for donor lung repair, reconditioning and regeneration, the perfusate should be modified to support the lung during extended EVLP. Human lung epithelial cells and pulmonary microvascular endothelial cells were cultured, and the effects of Steen solution (commonly used EVLP perfusate) on basic cellular function were tested. Steen solution was modified based on screening tests in cell culture, and further tested with an EVLP cell culture model, on apoptosis, GSH, HSP70, and IL-8 expression. Finally, a modified formula was tested on porcine EVLP. Physiological parameters of lung function, histology of lung tissue, and amino acid concentrations in EVLP perfusate were measured. Steen solution reduced cell confluence, induced apoptosis, and inhibited cell migration, compared to regular cell culture media. Adding L-alanyl-L-glutamine to Steen solution improved cell migration and decreased apoptosis. It also reduced cold preservation and warm perfusion-induced apoptosis, enhanced GSH and HSP70 production, and inhibited IL-8 expression on an EVLP cell culture model. L-alanyl-L-glutamine modified Steen solution supported porcine lungs on EVLP with significantly improved lung function, well-preserved histological structure, and significantly higher levels of multiple amino acids in EVLP perfusate. Adding L-alanyl-L-glutamine to perfusate may provide additional energy support, antioxidant, and cytoprotective effects to lung tissue. The pipeline developed herein, with cell culture, cell EVLP, and porcine EVLP models, can be used to further optimize perfusates to improve EVLP outcomes.

Temperature Dependence of Thermal Properties of Ex Vivo Porcine Heart and Lung in Hyperthermia and Ablative Temperature Ranges

This work proposes the characterization of the temperature dependence of the thermal properties of heart and lung tissues from room temperature up to > 90 °C. The thermal diffusivity (α), thermal conductivity (k), and volumetric heat capacity (Cv) of ex vivo porcine hearts and deflated lungs were measured with a dual-needle sensor technique. α and k associated with heart tissue remained almost constant until ~ 70 and ~ 80 °C, accordingly. Above ~ 80 °C, a more substantial variation in these thermal properties was registered: at 94 °C, α and k respectively experienced a 2.3- and 1.5- fold increase compared to their nominal values, showing average values of 0.346 mm2/s and 0.828 W/(m·K), accordingly. Conversely, Cv was almost constant until 55 °C and decreased afterward (e.g., Cv = 2.42 MJ/(m3·K) at 94 °C). Concerning the lung tissue, both its α and k were characterized by an exponential increase with temperature, showing a marked increment at supraphysiological and ablative temperatures (at 91 °C, α and k were equal to 2.120 mm2/s and 2.721 W/(m·K), respectively, i.e., 13.7- and 13.1-fold higher compared to their baseline values). Regression analysis was performed to attain the best-fit curves interpolating the measured data, thus providing models of the temperature dependence of the investigated properties. These models can be useful for increasing the accuracy of simulation-based preplanning frameworks of interventional thermal procedures, and the realization of tissue-mimicking materials.

Preparation, structure and antimicrobial properties of modified gelatin composite hydrogel from chicken, bovine and porcine lung

Preparation, structure and antimicrobial properties of modified gelatin composite hydrogel from chicken, bovine and porcine lung

Adsorption of porcine pulmonin can reduce the toxicity of gold nanorods and reduce lung injury

It is well known that porcine lung phospholipid injection (Curosurf) can effectively inhibit lung inflammation and reduce inflammatory cell damage to lung tissue, but whether it can reduce nanoparticles damage to lung remains to be seen. Poractant was co-incubated with gold nanorods as the model particle in this study to adsorb poractant on the surface of gold nanorods. Elisa kit was used to determine the adsorption degree of human lung surfactant associated protein poractant on the surface of gold nanorods. Gold nanorods adsorbed human lung surfactants associated proteins were evaluated further in vitro and in vivo. The toxicity of gold nanorods after absorption of porcine lung phospholipid was studied using human normal lung epithelial cells BEAS-2B; TNF- and IL-1 in serum were studied after absorption of porcine lung phospholipid using acute lung injury as a model. The results of the experiments show that adsorption of porcine lung phospholipids can reduce the toxicity of gold nanorods and the lung damage caused by gold nanorods.

Exploring pulmonary distribution of intratracheally instilled liquid foams in excised porcine lungs

The applicability of inhalation therapy to some severe pulmonary conditions is often compromised by limited delivery rates (i.e. total dose) and low deposition efficiencies in the respiratory tract, most notably in the deep pulmonary acinar airways. To circumvent such limitations, alternative therapeutic techniques have relied for instance on intratracheal liquid instillations for the delivery of high-dose therapies. Yet, a longstanding mechanistic challenge with such latter methods lies in delivering solutions homogeneously across the whole lungs, despite an inherent tendency of non-uniform spreading driven mainly by gravitational effects. Here, we hypothesize that the pulmonary distribution of instilled liquid solutions can be meaningfully improved by foaming the solution prior to its instillation, owing to the increased volume and the reduced gravitational bias of foams. As a proof-of-concept, we show in excised adult porcine lungs that liquid foams can lead to significant improvement in homogenous pulmonary distributions compared with traditional liquid instillations. Our ex-vivo results suggest that liquid foams can potentially offer an attractive novel pulmonary delivery modality with applications for high-dose regimens of respiratory therapeutics.

Efficacy of oxidized regenerated cellulose combined with fibrin glue in reducing pulmonary air leakage after segmentectomy in a porcine lung model.

Objectives: Pulmonary air leakage is a common complication following lung resection. We have designed a new method combining oxidized regenerated cellulose and fibrin glue to cover the intersegmental plane in clinical lung segmentectomy to prevent postoperative air leakage. In this study, an excised porcine lung segmentectomy model was created to validate its adhesive strength and effect on reducing air leakage. Methods: In the pre-experiment, six different larger lung segments were separated using electrocautery on the fresh isolated porcine lungs (n = 5 in each group). The air leakage degree and operation time of the lung segments were comprehensively evaluated to select the most suitable target segment for establishing the ex vivo porcine lung segmentectomy models. In the experiment, according to the different materials covered on the intersegmental plane, these models were randomly divided into four groups: group A used fibrin glue and oxidized regenerated cellulose (ORC) mesh (n = 20); group B used fibrin glue and polyglycolic acid (PGA) sheet (n = 20); group C used fibrin glue (n = 20); group D was the blank control group (n = 20). The minimum air leakage pressure (MALP) of the selected target segment in each group was measured using a stepwise increase of airway pressure, and histological assessment was performed on the sealed area samples from the four groups. Results: The operation time of the a segment of the right cranial lobe (R1a) was shorter than that of other segments (p < 0.05), and there was no significant difference in the air leakage pressures between the six isolated segments (p = 0.76); thus, R1a was chosen for segmentectomy. In addition, the MALP was significantly higher in group A (41.8 ± 4.5 cmH2O) than in groups C (28.1 ± 2.3 cmH2O) and D (17.3 ± 1.2 cmH2O) (both p < 0.001). The MALP of group B (69.5 ± 5.2 cmH2O) was significantly higher than that of group A (p < 0.001), whereas that of group C was significantly higher than that of group D (p < 0.001). Histological examination confirmed that the combined use of fibrin glue and ORC or PGA patch adhered more firmly to the intersegmental plane than that of fibrin glue alone, although some gaps could be seen between the fibrin glue and the surface of the lung segments in group C. Conclusion: The application of ORC combined with fibrin glue on the intersegmental plane has a good sealing performance in the ex vivo porcine lung segmentectomy model, suggesting that ORC may be an effective alternative material to replace PGA sheet to combine with fibrin glue for preventing air leakage after segmentectomy.

Microwave ablation of the lung: Comparison of 19G with 14G and 16G microwave antennas in ex vivo porcine lung.

Percutaneous image-guided thermal ablation has an increasing role in the treatment of primary and metastatic lung tumors. Although microwave ablation (MWA) has emerged advantageous as a new ablation technology, more research is needed to improve it. This study aims to investigate the ablation zone of three microwave antennas in ex vivo porcine lung. In the ex vivo standard model and porcine lung model, MWA was performed in three power output settings (50 W, 60 W, and 70 W) for 3, 6, 9, and 12 min using three microwave antennas, with outer diameter of 1.03 mm (19G), 1.6 mm (16G), and 2.0 mm (14G). A total of 108 and 216 sessions were performed (3 or 6 sessions per time setting with the 14G, 16G, and 19G microwave antennas). After the MWA was complete, we evaluated the shape and extent of the coagulation zone and measured the maximum long-axis (along the needle axis; length [L]) and maximum short-axis (perpendicular to the needle; diameter [D]) of the ablation zones using a ruler; subsequently, the sphericity index (L/D) was calculated. The sphericity index can be simplified as long-axis/short-axis. In the ex vivo standard model study, the long- and short-axis diameters and sphericity indices were not statistically different between the 14G, 16G, and 19G groups. In the ex vivo porcine lung study, the long- and short-axis diameters did not differ statistically between the 14G, 16G, and 19G groups (P < 0.05 each). The sphericity index for the 19G microwave antenna was higher than the sphericity indices for the 14G and 16G microwave antennas (P < 0.05); however, the index for the 14G microwave antenna was not statistically different than that for the 16G microwave antenna (P > 0.05). The ablation zone of the 19G antenna was the same as those of the 14G and 16G antennas in vitro. Thus, the 19G antenna may reduce the incidence of complications in lung tumor ablation.

Vision-Kinematics Interaction for Robotic-Assisted Bronchoscopy Navigation.

Endobronchial intervention is increasingly used as a minimally invasive means for the treatment of pulmonary diseases. In order to acquire the position of bronchoscopy, vision-based localization approaches are clinically preferable but are sensitive to visual variations. The static nature of pre-operative planning makes mapping of intraoperative anatomical features challenging for learning-based methods using visual features alone. In this work, we propose a robust navigation framework based on Vision Kinematic Interaction (VKI) for monocular bronchoscopic videos. To address visual-imbalance between the virtual and real views of bronchoscopy images, a Visual Similarity Network (VSN) is proposed to extract domain-invariant features to represent the lumen structure from endoscopic views, as well as domain-specific features to characterize the surface texture and visual artefacts. To improve the robustness of online estimation of camera pose, we also introduce a Kinematic Refinement Network (KRN) that allows progressive refinement of camera pose estimation based on network prediction and robot control signals. The accuracy of camera localization is validated on phantom and porcine lung datasets from a robotically controlled endobronchial intervention system, with both quantitative and qualitative results demonstrating the performance of the techniques. Results show that the features extracted by the proposed method can preserve the structural information of small airways in the presence of large visual variations along with the much-improved camera localization accuracy. The absolute trajectory errors (ATE) on phantom data and porcine data are 8.01 mm and 8.62 mm respectively.

Distribution, composition, and activity of airway-associated adipose tissue in the porcine lung.

Patients with comorbid asthma-obesity experience greater disease severity and are less responsive to therapy. We have previously reported adipose tissue within the airway wall that positively correlated with body mass index. Accumulation of biologically active adipose tissue may result in the local release of adipokines and disrupt large and small airway function depending on its anatomical distribution. This study therefore characterized airway-associated adipose tissue distribution, lipid composition, and adipokine activity in a porcine model. Airway segments were systematically dissected from different locations of the bronchial tree in inflation-fixed lungs. Cryosections were stained with hematoxylin and eosin (H&E) for airway morphology, oil red O to distinguish adipose tissue, and Nile blue A for lipid subtype delineation. Excised airway-associated adipose tissue was cultured for 72 h to quantify adipokine release using immunoassays. Results showed that airway-associated adipose tissue extended throughout the bronchial tree and occupied an area proportionally similar to airway smooth muscle within the wall area. Lipid composition consisted of pure neutral lipids (61.7 ± 3.5%), a mixture of neutral and acidic lipids (36.3 ± 3.4%), or pure acidic lipids (2.0 ± 0.8%). Following tissue culture, there was rapid release of IFN-γ, IL-1β, and TNF-α at 12 h. Maximum IL-4 and IL-10 release was at 24 and 48 h, and peak leptin release occurred between 48 and 72 h. These data extend previous findings and demonstrate that airway-associated adipose tissue is prevalent and biologically active within the bronchial tree, providing a local source of adipokines that may be a contributing factor in airway disease.

Establishment and characterization of the immortalized porcine lung-derived mononuclear phagocyte cell line.

Mononuclear phagocytes (MNP), including monocytes, dendritic cells (DC), and macrophages, play critical roles in innate immunity. MNP are abundant in the lungs and contribute to host defense against airborne agents and pulmonary immune homeostasis. In this study, we isolated porcine lung-derived MNP (PLuM) from primary cultures of parenchymal lung cells and then immortalized them by transferring the SV40 large T antigen gene and porcine telomerase reverse transcriptase gene using lentiviral vectors. The established cell line, immortalized PLuM (IPLuM), expressed DC/macrophage markers; i.e., CD163, CD172a, and major histocompatibility complex class II, whereas they did not express a porcine monocyte-specific marker, CD52. The expression patterns of these cell surface markers indicate that IPLuM originate from the DC/macrophage lineage rather than the monocyte lineage. The bacterial cell wall components muramyl dipeptide and lipopolysaccharide induced the production of the interleukin-1 family of pro-inflammatory cytokines in IPLuM. Phagocytotic activity was also detected by time-lapse fluorescence imaging of live cells when IPLuM were cultured in the presence of pHrodo dye-conjugated E. coli BioParticles. It is worth noting that IPLuM are susceptible to African swine fever virus infection and support the virus' efficient replication in vitro. Taken together, the IPLuM cell line may be a useful model for investigating host-agent interactions in the respiratory microenvironments of the porcine lung.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n=18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6h whereas two reference lungs failed before elective termination at 4h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p=.007; IgG p<.001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p=.007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p=.023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p=.007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p=.003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.