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Transplantation of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. In previous studies we demonstrated that conventional frozen cryopreservation (FC) is accompanied by serious alterations of extracellular matrix (ECM) structures. As the main culprit of the observed damages ice crystal formation was identified. Objective of this study was the application principles of cryoprotection as observed in nature, occurring in animals or plants, for ice-free cryopreservation (IFC) of heart valves. Using IFC, valves were processed and stored above the glass transition temperature of the cryoprotectant formulation (-124 degrees C) at -80 degrees C to avoid any ice formation, tissue-glass cracking and preserving ECM. After implantation in the orthotopic pulmonary position in sheep, we demonstrate that IFC resulted in cell free matrices, while maintaining crucial ECM-components such as elastin and collagen, translating into superior hemodynamics. In contrast, we reveal that FC valves showed ECM damage that was not restored in vivo, and T-cell inflammation of the stroma with significant leaflet thickening. Compared to currently applied FC practice IFC also reduced infrastructural needs for preservation, storage and shipping. These results have important implications for clinical valve transplantation including the promise of better long-term function and lower costs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Objective: Optimal preservation of postischemic graft function is essential in lung transplantation. Antegrade flush perfusion with modified Euro-Collins solution represents the standard technique worldwide. However, growing evidence suggests the superiority of extracellular-type Perfadex solution (Vitrolife AB, Gothenburg, Germany) over Euro-Collins solution. During ischemia and reperfusion, endogenous pulmonary nitric oxide synthesis is decreased, and therefore therapeutic stimulation of the nitric oxide pathway might be beneficial in ameliorating ischemia-reperfusion damage. However, research mainly focuses on nitric oxide supplementation of intracellular solutions, and no studies exist in which the effect of nitroglycerin on Perfadex preservation quality is evaluated., Methods: Eight rat lungs each were preserved with Perfadex solution with or without nitroglycerin (0.1 mg/mL) and compared with low-potassium Euro-Collins solution. Postischemic lungs were reventilated and reperfused, and oxygenation capacity, pulmonary vascular resistance, and peak inspiratory pressures were monitored continuously. Stereological analysis was used for evaluation of pulmonary edema and assessment of the vasculature. Statistics were performed by using different analysis of variance models., Results: The oxygenation capacity of the Perfadex-preserved groups was higher compared with that of the low-potassium Euro-Collins solution group (P <.03). By using nitroglycerin, flush-perfusion time was reduced, and Perfadex solution with nitroglycerin-protected lungs showed superior oxygenation capacity compared with that seen in Perfadex solution-protected organs (P <.01). Furthermore, pulmonary vascular resistance and peak inspiratory pressures were improved in the nitroglycerin group (P <.01). Stereology revealed comparable intrapulmonary edema between groups and a trend toward less vasoconstricted vasculature in Perfadex with nitroglycerin-protected lungs., Conclusions: Perfadex solution provides superior lung preservation in terms of postischemic oxygenation capacity than Euro-Collins solution. Supplementation of the nitric oxide pathway by nitroglycerin further enhances functional outcome of Perfadex-preserved organs and might be an easily applicable tool in clinical lung transplantation.

Objective: Extracorporeal lung-perfusion models are widely used to evaluate pulmonary preservation techniques and reperfusion injury. However, these models mainly depend on nonpulsatile flow, which is not physiological and can subsequently lead to pulmonary edema. Observation in a standardized setting and reliability of functional and structural data assessment are therefore limited. To overcome these limitations we developed a new extracorporeal large animal lung perfusion model utilizing pulsatile flow to perfuse the pulmonary vasculature., Methods: Lungs of juvenile domestic pigs were in situ preserved with 2 liters Perfadex and stored for 3 h at 10 degrees C. Thereafter, reperfusion of the lung was performed in an extracorporeal blood perfusion circuit employing either a modified roller pump with pulsatile module (300 ml/min; pulsation rate 90/min) or a standardized roller pump with continuous flow (30 ml/min). Ventilation was performed with physiologic room air (350 ml; 16/min) for 1 h. Pulsatile and nonpulsatile perfusion was performed in 2 groups (group NP: nonpulsatile; group P: pulsatile flow, n = 7) during reperfusion. Peak inspiratory pressure (PIP), mean pulmonary artery pressure (PAP), and oxygenation capacity (DeltaPO(2)) were continuously measured. For control of the effectiveness of the pulsatile perfusion pressure waveforms were obtained directly from the native pulmonary artery of both groups. Malondialdehyde (MDA) as a parameter for lipid peroxidation and endothelial cell damage was assessed at 10, 30 and 50 min reperfusion. At the end of the study, pulmonary water content was assessed by means of wet-to-dry ratio (W/D ratio). The tissue was further processed for microscopic analysis., Results: PIP increased significantly in both groups during reperfusion. Mean PAP in both groups increased to 60 mm Hg after 20 min followed by a decrease after 60 min to 40 mm Hg. Pressure waveforms of the pulmonary artery showed sufficient pulsatility in the pulmonary vasculature with a systolic/diastolic pressure difference of 15 mm Hg whereas the pressure difference was 3-5 mm Hg in the nonpulsatile group. DeltaPO(2) was stable in groups NP and P during reperfusion (30 min: NP: 66.4 (62.2-88) mm Hg; P: 74.8 (65-81.7) mm Hg) without any statistically significant differences between the groups. MDA in group NP decreased over the reperfusion period from 6.2 (3.3-6.3) microM at 10 min to 5.2 (3.2-6.1) microM at 50 min, whereas in group P the level increased and was significantly higher after 50 min reperfusion compared to group NP [6.6 (6.1-9.2) microM at 50 min; p = 0.016]. W/D ratio was 6.7 (6.3-7.0) in group NP and 6.8 (6.3-7.6) in group P. Light microscopy evaluation showed no differences between both groups regarding severity of intra-alveolar and interstitial edema and numbers of intra-alveolar, intracapillary and interstitial granulocytes., Conclusion: Although effective pulsatile perfusion of the pulmonary vasculature was achieved by means of a modified roller pump, this measure obviously did not improve functional parameters nor did it significantly reduce the edema formation after 3 h ischemia in this extracorporeal lung perfusion model. The use of pulsatile perfusion is therefore not mandatory in the extracorporeal setting of a large animal lung perfusion model., (Copyright 2002 S. Karger AG, Basel)

Background: Extracellular preservation solutions utilizing high molecular agents can reduce intracellular edema during ischemia/reperfusion in lung transplantation. A solution of 40,000 dalton molecular weight (DMW) has already been clinically established (Perfadex). However, it is unclear whether dextrans of this particular size represent the optimal additive for lung preservation solutions., Materials and Methods: In a new ex vivo porcine lung model, lungs were each preserved with low-potassium solutions containing 5% dextran with 90,000 DMW (Dex 90) and 160,000 DMW (Dex 160) and with Perfadex (40,000 DMW). After 24 h of cold ischemia, reperfusion was performed employing a roller pump with a pulsatile module. Lungs were perfused with deoxygenated perfusate and ventilated with room air. The oxygenation capacity (Delta pO(2)), peak inspiratory pressure (PIP), and mean pulmonary artery pressure (PAP) were monitored for 60 min. Net weight gain (NWG) and wet-to-dry ratio (W/D ratio) were determined. Free-radical generation was assessed by measuring malondialdehyde (MDA) at 10, 30, and 50 min., Results: PIP and PAP increased in all groups significantly during reperfusion. However, Dex 160-perfused lungs exhibited significantly higher values than those with Dex 90 and Perfadex. Perfadex showed the highest Delta pO(2) throughout the entire reperfusion, while Delta pO(2) was slightly reduced in Dex 160 and significantly lower in Dex 90. In Perfadex the lowest water content was observed assessed by NWG and W/D ratio. The highest MDA values were observed in Dex 90, followed by Dex 160, while the lowest values were seen in Perfadex., Conclusions: Preservation of the lung with Perfadex exhibited superior postischemic function in contrast to preservation solutions containing dextrans with a higher molecular weight.

Ischemia and reperfusion (I/R) result in surfactant dysfunction. Whether the impairment of surfactant is a consequence or a cause of intraalveolar edema formation is still unknown. The cumulative effects of lung perfusion, ischemic storage, and subsequent reperfusion on surfactant ultrastructure and pulmonary function were studied in a rat isolated perfused lung model. The left lungs were fixed for electron microscopy by vascular perfusion either immediately after excision (control; n = 5) or after perfusion with modified Euro-Collins solution (EC), storage for 2 h at 4 degrees C in EC, and reperfusion for 40 min (n = 5). A stereological approach was chosen to discriminate between intraalveolar surfactant subtypes of edematous regions and regions free of edema. Intraalveolar edema seen after I/R in the EC group occupied 36 +/- 6% (mean +/- SEM) of the gas exchange region as compared with control lungs (1 +/- 1%; p = 0.008). Relative intraalveolar surfactant composition showed a decrease in surface active tubular myelin (3 +/- 1 versus 12 +/- 0%; p = 0.008) and an increase in inactive unilamellar forms (83 +/- 2 versus 64 +/- 5%; p = 0.008) in the EC group. These changes occurred both in edematous (tubular myelin, 3 +/- 1%; unilamellar forms, 88 +/- 6%) and in nonedematous regions (tubular myelin, 4 +/- 3%; unilamellar forms, 77 +/- 5%). The ultrastructural changes in surfactant were associated with an increase in peak inspiratory pressure during reperfusion. In conclusion, surfactant alterations seen after I/R are not directly related to the presence of edema fluid in the alveoli. Disturbances in intraalveolar surfactant after I/R are not merely the result of inactivation due to plasma protein leakage but may instead be responsible for an increased permeability of the blood-air barrier, resulting in a vicious cycle of intraalveolar edema formation and progressing surfactant impairment.

Early graft dysfunction after lung transplantation is a significant and unpredictable problem. Our study aimed at a detailed investigation of structure-function correlations in a rat isolated heart-lung model ofischemia/ reperfusion injury. Variable degrees of injury were induced by preservation with potassium-modified Euro-Collins solutions, 2 hr of cold ischemia, and 40 min of reperfusion. Pulmonary artery pressure (Ppa), pulmonary vascular resistance (PVR), peak inspiratory pressure (PIP), and perfusate gases (deltaPO2, deltaPCO2) were recorded during reperfusion. Right lungs were used to calculate W/D-weight ratios. Nineteen experimental and six control left lungs were fixed for light and electron microscopy by vascular perfusion. Systematic random samples were analyzed by stereology to determine absolute and relative volumes of lung structures, the amount of interstitial and intraalveolar edema, and the extent of epithelial injury. Lectin- and immunohistochemistry using established epithelial cell markers were performed in three animals per group to reveal sites of severe focal damage. Experimental lungs showed a wide range in severity of ischemia/ reperfusion injury. Intraalveolar edema fluid amounted to 77-909 mm3 with a mean of 448+/-250 mm3 as compared with 22+/-22 mm3 in control lungs (P<0.001). Perfusate oxygenation (deltaPO2) decreased from 30.5+/-15.2 to 21.7+/-15.2 mm Hg (P=0.05) recorded after 5 and 40 minutes of reperfusion. In experimental lungs, a surface fraction of 1% to 58% of total type I pneumocyte surface was damaged. Intraalveolar edema per gas exchange region (Vv ape,P) and deltaPO2 were related according to deltaPO2 = 96 - 60 x log10(Vv ape,P) [mm Hg]. The extent of epithelial injury did not correlate with deltaPO2 nor with intraalveolar edema, but increased significantly with PVR. Lectin- and immunohistochemistry revealed focal severe damage to the alveolar epithelium at the border of perivascular cuffs.

Background: Low-potassium solutions have been shown to improve lung preservation. The optimal potassium concentration, however, has not been investigated systematically. The purpose of this study was to evaluate the effect of solutions with different potassium concentrations on functional and structural preservation after flush-perfusion and ischemia. We used our established extracorporeal working heart-lung model and a modification of this model with isolated pulmonary perfusion at defined flow rates., Methods: In two sets of experiments 42 rat heart-lung blocks (experiment I and II: n=7/group) were used. Lungs were flush-preserved with 20 ml Euro-Collins solution (EC115; K+ 115 mmol/L), potassium-reduced Euro-Collins solution (EC40; K+ 40 mmol/L), or low-potassium Euro-Collins solution (EC10; K+ 10 mmol/L) and stored for 2 hours at 10 degrees C. Reperfusion was performed for 40 minutes with Krebs-Henseleit solution containing washed bovine red blood cells (38%) while the lungs were ventilated with room air. In experiment I pulsatile perfusion of the lungs was achieved by the working right side of the heart. In experiment II lungs were perfused at defined flow rates by a roller pump. Postischemic function was assessed by means of oxygenation capacity and pulmonary vascular resistance. The degree of structural damage to the air-blood barrier was assessed by quantitative stereologic light and electron microscopic evaluation., Results: In both experiments after 40 minutes reperfusion oxygenation capacity was significantly higher in EC40 than in EC115 and EC10, whereas pulmonary vascular resistance was significantly higher in EC115 than in EC40 and EC10. Quantitative histologic examination showed surprisingly modest damage to the endothelial side of the air-blood barrier but a considerable degree of damage to the epithelium in both experiments. The alterations in the pump-perfused isolated lung experiments exceeded those of the pulsatile perfused heart-lung experiments. The comparative analysis of the study groups revealed a minor degree of epithelial swelling and fragmentation in EC40 than in EC115 and EC10, respectively., Conclusions: The results obtained with two modifications of an extracorporeal model indicate that flush perfusion of the lung with a potassium-reduced solution results in better functional and structural preservation than flush perfusion with either high- or low-potassium solutions. The optimum may lie in the vicinity of 40 mmol/L. Further studies are necessary to verify these initial findings.

The optimal temperature of the pulmonary flush perfusate is still a matter of controversy. At present, a temperature of 10 degrees C is favored. This study deals with the structural and functional impact of different perfusate temperatures on lung preservation. In an extracorporeal rat heart-lung model lungs were preserved with Perfadex solution of 4, 15 and 25 degrees C and submitted to 2 h ischemia. Heart-lung blocks harvested from male rats were perfused with Krebs-Henseleit solution and ventilated with room air. Lungs were perfused with deoxygenated perfusate via the working right ventricle while the coronary arteries were retrogradely perfused with oxygenated perfusate. Oxygenation capacity (dPO2), peak inspiratory pressure (PIP) and pulmonary vascular resistance were measured. After establishment of baseline functional parameters hearts were arrested with 10 ml St. Thomas cardioplegia and lungs were flushed with 20 ml Perfadex solution. The heart-lung block was then stored for 2 h at 10 degrees C. Reperfusion was performed thereafter under the same conditions. At the end of the trial the lung tissue water was measured by the wet/dry ratio. The perfusion time of the groups flushed with 15 or 25 degrees C perfusate was significantly lower than that of the 4 degrees C group. After 20 min reperfusion dPO2 of the groups flushed with 15 or 25 degrees C was superior to those submitted to a 4 degrees C flush perfusion. PIP was significantly lower in the 15 degrees C group than in the 4 and 25 degrees C groups. The wet/dry ratio revealed the smallest water content in the 15 degrees C group. We conclude that the post-ischemic lung function is dependent on the temperature of the flush perfusate. Among the tested temperatures, perfusion at 15 degrees C showed the best results. The optimum may therefore lie in this range.

In recent years several potassium-reduced solutions have been developed for improvement of pulmonary preservation. A comparison of these solutions in a single study, however, has not yet been performed. In an extracorporeal working rat heart-lung model (n = 7/group) lungs were preserved with 20 ml Euro-Collins (EC), low potassium EC (LPEC), low potassium 2% dextran (LPD), ET-Kyoto (ETK) or low potassium 5% dextran (Perfadex) solution, while hearts were arrested with 10 ml St. Thomas' cardioplegia. Lungs of controls were not perfused. The heart-lung blocks were stored for 2 h at 10 degrees C. Thereafter, heart-lung blocks were extracorporeally perfused with Krebs-Henseleit solution with washed bovine red blood cells. Coronaries were perfused with oxygenated perfusate. Lungs were perfused via the working right ventricle with deoxygenated perfusate and ventilated with room air. Oxygenation capacity (dPO2) and pulmonary vascular resistance (PVR) were measured. Reperfusion/ventilation was performed for 40 min. At the end of the experiment the wet to dry (W/D) ratio of lungs and light microscopic assessment of the degree of edema (0-4) were performed. All potassium-reduced solutions showed superior dPO2 and a lower PVR than EC and controls while LPEC exhibited the most stable dPO2 and lowest PVR after 30 min reperfusion. The W/D ratio of all potassium-reduced groups was lower than the ratio of EC and controls. In LPEC, ETK and Perfadex the least degree of edema was noted. All solutions used in this study are superior to regular EC. However, when compared directly, LPEC perfused lungs showed better functional preservation than all other alternative solutions.

Crystalloid perfusates commonly are utilized for lung preservation in extracorporeal small animal lung models. However, the function of these grafts is limited. In a new ex-vivo rat heart-lung model the role of red blood cells added to the crystalloid perfusate was investigated. Heart-lung blocks were rapidly excised (n = 9, each group) and the blocks were connected to the extracorporeal perfusion circuit using Krebs-Henseleit solution (KH) or KH with washed bovine red blood cells (hematocrit 38%) (KHRBC). The lungs were ventilated with room air. The coronary system was perfused via the aortic root with oxygenated perfusate. The lungs were perfused via the right ventricle with deoxygenated perfusate (PO2 15 mm Hg). Venoarterial improvement of oxygenation, pulmonary vascular resistance (PVR), and peak inspiratory pressure (PIP) were continuously monitored for 50 min. At the end of the experiment the wet/dry (W/D) ratio was determined using the mediastinal lung lobe while the remaining lung was used for light microscopic (LM) evaluation. After 30 min of perfusion, lung function was significantly better in the KHRBC group (PVR (KH): 752 +/- 193 dyn*sec*cm-5; (KHRBC): 279 +/- 48 dyn*sec*cm-5; PIP (KH): 31 +/- 3.2 mm Hg; (KHRBC): 25.8 +/- 1.9 mm Hg). In addition, lungs perfused with KHRBC showed significantly less edema than those perfused with KH only (W/D ratio (KH): 7.8 +/- 1.2; (KHRBC) 5.1 +/- 0.6; LM (KH): 3.5 +/- 0.9; (KHRBC): 2.3 +/- 0.8). The use of red blood cells in KH perfusate improved functional and structural integrity.(ABSTRACT TRUNCATED AT 250 WORDS)