Cold Preservation Research Articles

Lung Lavage and Surfactant Replacement During Ex Vivo Lung Perfusion for Treatment of Gastric Acid Aspiration–Induced Donor Lung Injury

Ex vivo lung perfusion (EVLP) provides opportunities to treat injured donor lungs before transplantation. We investigated whether lung lavage, to eliminate inflammatory inhibitory components, followed by exogenous surfactant replacement, could aid lung recovery and improve post-transplant lung function after gastric aspiration injury. Gastric acid aspiration was induced in donor pigs, which were ventilated for 6 hours to develop lung injury. After retrieval and 10 hours of cold preservation, EVLP was performed for 6 hours. The lungs were randomly divided into 4 groups (n = 5, each): (1) no treatment (control), (2) lung lavage, (3) surfactant administration, and (4) lung lavage, followed by surfactant administration. After another 2-hour period of cold preservation, the left lung was transplanted and reperfused for 4 hours. Physiologic lung function significantly improved after surfactant administration during EVLP. The EVLP perfusate from the lavage + surfactant group showed significantly lower levels of interleukin (IL)-1β, IL-6, IL-8, and secretory phospholipase A2. Total phosphatidylcholine was increased, and minimum surface tension was recovered to normal levels (≤5 mN/m) in the bronchioalveolar fluid after surfactant administration. Lysophosphatidylcholine in bronchioalveolar fluid was significantly lower in the lavage + surfactant group than in the surfactant group. Post-transplant lung function was significantly better in the lavage + surfactant group compared with all other groups. Lung lavage, followed by surfactant replacement during EVLP, reduced inflammatory mediators and prevented hydrolysis of phosphatidylcholine, which contributed to the superior post-transplant function in donor lungs with aspiration injury.

Machine Perfusion of Porcine Livers with Oxygen-Carrying Solution Results in Reprogramming of Dynamic Inflammation Networks.

Background: Ex vivo machine perfusion (MP) can better preserve organs for transplantation. We have recently reported on the first application of an MP protocol in which liver allografts were fully oxygenated, under dual pressures and subnormothermic conditions, with a new hemoglobin-based oxygen carrier (HBOC) solution specifically developed for ex vivo utilization. In those studies, MP improved organ function post-operatively and reduced inflammation in porcine livers. Herein, we sought to refine our knowledge regarding the impact of MP by defining dynamic networks of inflammation in both tissue and perfusate.Methods: Porcine liver allografts were preserved either with MP (n = 6) or with cold static preservation (CSP; n = 6), then transplanted orthotopically after 9 h of preservation. Fourteen inflammatory mediators were measured in both tissue and perfusate during liver preservation at multiple time points, and analyzed using Dynamic Bayesian Network (DyBN) inference to define feedback interactions, as well as Dynamic Network Analysis (DyNA) to define the time-dependent development of inflammation networks.Results: Network analyses of tissue and perfusate suggested an NLRP3 inflammasome-regulated response in both treatment groups, driven by the pro-inflammatory cytokine interleukin (IL)-18 and the anti-inflammatory mediator IL-1 receptor antagonist (IL-1RA). Both DyBN and DyNA suggested a reduced role of IL-18 and increased role of IL-1RA with MP, along with increased liver damage with CSP. DyNA also suggested divergent progression of responses over the 9 h preservation time, with CSP leading to a stable pattern of IL-18-induced liver damage and MP leading to a resolution of the pro-inflammatory response. These results were consistent with prior clinical, biochemical, and histological findings after liver transplantation.Conclusion: Our results suggest that analysis of dynamic inflammation networks in the setting of liver preservation may identify novel diagnostic and therapeutic modalities.

Retained fertilizing capability in cryopreserved feline spermatozoa.

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

Food Quality Induces a Miscible Disease with Relevance to Alzheimer’s Disease and Neurological Diseases

Food and nutrition guidelines for the handling and processing of fresh fruit, bread, and vegetables are essential and fresh produce may require cold preservation procedures to prevent minimal bacterial and fungi contamination of food. Bacterial lipopolysaccharides (LPS) corrupt lipoprotein and amyloid beta (Aβ) metabolism in diabetes, Alzheimer’s disease (AD) and various neurological diseases. In the developing world the increased plasma LPS levels induce non-alcoholic fatty liver diseases and interfere with albumin and Aβ interactions with spontaneous Aβ oligomer formation in the cerebrospinal fluid and brain that leads to neuron apoptosis by inactivation of Starling’s equation that is responsible for the maintenance of hydrostatic and oncotic pressure with relevance to fluid balance. In the developing world increased levels of LPS, mycotoxin and xenobiotics lead to irreversible neurological diseases by inhibition of Starling’s equation for maintenance of oncotic/osmotic pressure that lead to neuron senescence or apoptosis. In the developed world nutrigenomic diets are required that prevent Sirtuin 1 gene repression and maintain neuron survival that links the brain and peripheral hepatic monomer Aβ metabolism. The maintenance of blood-cerebrospinal fluid capillary transport of albumin and monomer Aβ is relevant to stabilization of neurons not only in Alzheimer’s disease but also in Type 3 diabetes and various neurological diseases. Healthy diets reverse the inhibition of brain to peripheral Aβ transport that is sensitive to Starling’s equation for regulation of central nervous system hydrostatic and oncotic pressure with the prevention of diabetes, various neurological diseases and Alzheimer’s disease.

Effects of Nitrite Addition to IGL-1 Solution on Rat Liver Preservation.

BACKGROUND The ability of nitrite to provide protection following ischemia/reperfusion (I/R) has been demonstrated, but its mechanism is still poorly understood. This study aimed to determine the optimal nitrite concentration to add into Institut Georges Lopez (IGL-1) storage solution and to assess its effect on antioxidant enzymes and autophagy. MATERIAL AND METHODS Livers from Sprague-Dawley rats were conserved in IGL-1 for 24 hours at 4°C or in IGL-1 enriched with nitrite at 50, 500 and 1,000 nM, respectively, before being perfused ex-vivo at 37 °C for 120 minutes. Sham livers were perfused ex vivo without cold preservation. RESULTS All biological and functional parameters of the preserved livers were significantly impaired as compared to shams. Interestingly, the supplementation of nitrite to IGL-1 protected the liver from I/R injury. Among the doses of nitrite evaluated, the 50 nM was proved efficient: it significantly reduced cytolysis, mitochondrial damage, and lipid peroxidation, and enhanced antioxidant enzyme activity (superoxide dismutase, catalase, and glutathione peroxidase activity) and hepatic function parameters (portal resistance, bile flow, and bromosulfophthalein clearance). In addition, increased levels of the autophagy parameters were observed when 50 nM of nitrite were added to IGL-1 solution, but this effect disappeared completely with higher concentrations of nitrite. CONCLUSIONS It seems that 50 nM of nitrite added to IGL-1 is the optimal concentration able to maintain cell integrity and hepatic function through autophagy induction and oxidative stress prevention.

Donation After Circulatory Death Lungs Transplantable Up to Six Hours After Ex Vivo Lung Perfusion

Despite the critical need for donor lungs, logistic and geographic barriers hinder lung utilization. We hypothesized that lungs donated after circulatory death subjected to 6 hours of cold preservation after exvivo lung perfusion (EVLP) would have similar outcomes after transplantation as lungs transplanted immediately after EVLP, and that both would perform superiorly compared with lungs transplanted immediately after procurement. Donor porcine lungs were procured after circulatory death and 15 minutes of warm ischemia. Three groups (n= 5 per group) were randomized: immediate left lung transplantation (Immediate group), EVLP for 4 hours followed by transplantation (EVLP group), or EVLP for 4 hours followed by 6 hours of cold preservation followed by transplantation (EVLP+Cold group). Lungs were reperfused for 2 hours before obtaining pulmonary vein samples for partial pressure of oxygen/fraction of inspired oxygen ratio calculations, airway pressures for compliance measurements, and wet/dry weight ratios. The partial pressure of oxygen/fraction of inspired oxygen ratios in the EVLP and EVLP+Cold groups were significantly improved compared with those in the Immediate group (429.7 ± 51.8 and 436.7 ± 48.2 versus 117.4 ± 22.9 mm Hg, respectively). In addition, dynamic compliance was significantly improved in the EVLP and EVLP+Cold groups compared with immediate group (26.2 ± 4.2 and 27.9 ± 3.5 versus 11.1 ± 2.4 mL/cmH2O, respectively). There were no differences in oxygenation capacity or dynamic compliance between the EVLP and EVLP+Cold groups. Inflammatory cytokine levels were significantly lower in the EVLP and EVLP+Cold groups. Lungs donated after circulatory death can be successfully transplanted as much as 6 hours after EVLP. Cold preservation of lungs after exvivo assessment and rehabilitation may improve organ allocation, even to distant recipients, without compromising allograft function.

Extending Vascularized Composite Tissue Survival Using Near-Normothermic Ex Situ Perfusion

Objective/Hypothesis: Currently, vascularized composite allografts (VCAs) are cold preserved (4°C) until transplantation. This process is time limited, as the tissue has to be revascularized within 4 to 6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation. This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury. Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours. In this study, we aimed to test this system on human forearm allografts. Material and Methods: Five human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature-controlled (30°C-33°C) ex situ perfusion system for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4 to 6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Results: Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 93 ± 2 mm Hg, perfusion flow 310 ± 20 mL/min (~6%-8% of the donor’s estimated cardiac output), and vascular resistance 153 ± 16 mm Hg/mL/min. Perfusate had an average pH of 7.43 ± 0.04, pCO2 32 ± 1 mm Hg, pO2 317 ± 18 mm Hg, and Hb 4.8 ± 0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment. Hematoxylin-eosin staining showed mild fatty infiltration on some myocytes at 24 hours. There was minimal change in fiber size, likely due to variation in age and gender between donors. Muscle architecture was preserved at the end of 24 hours perfusion. Conclusions: All limbs remained viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation. While no assumptions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area. It also has the potential to circumvent complications associated with cold preservation.

Numerical Simulation of Cold Storage and Preservation of Hanfu Apple

To solve the decay phenomenon of Hanfu apples that occurs during storage and obtain the best environmental parameters for preserving Hanfu apples using Fluent. Methods: Preservation storage was designed by analyzing the characteristics of Hanfu-apple storage and the natural environment in northeast China. The boundary conditions and simulation of a preservation model were established. Results: Compared with the initial values, the hardness decreased by 1.95 kg/cm² , the sugar-acid ratio decreased by 20.43, and weight-loss rate was 3.98%. Conclusion: The results for the temperature field and velocity field of the empty storage were analyzed using Fluent. Hanfu apples can maintain good quality during the period of storage, according to our analysis of the Hanfu-apple hardness, weight loss, and change in the sugar-acid ratio during the 90-day preservation period.

Effect of Cold Preservation on Chronic Rejection in a Rat Hindlimb Transplantation Model.

Previous studies on solid organ transplantation have shown that cold ischemia contributes to the development of chronic allograft vasculopathy. The authors evaluated the effect of cold ischemia on the development of chronic rejection in vascularized composite allotransplantation. Thirty rat hindlimbs were transplanted and divided into two experimental groups: immediate transplantation and transplantation after 7 hours of cold ischemia. The animals received daily low-dose immunosuppression with cyclosporine A for 2 months. Intimal proliferation, arterial permeability rate, leukocyte infiltration, and tissue fibrosis were assessed. The CD3, CD4, CD8, CD20, and CD68 cells per microscopic field (200×) were counted, and C4d deposition was investigated. Cytokine RNA analysis was performed to measure tumor necrosis factor-α, interleukin-6, and interleukin-10 levels. Significant differences were found in the intimal proliferation and arterial permeability rate between the two groups (p = 0.004). The arterial permeability rate worsened in the most distal and small vessels (p = 0.047). The numbers of CD3, CD8, CD20, and CD68 were also statistically higher in the cold ischemia group (p < 0.05, all levels). A trend toward significance was observed with C4d deposition (p = 0.059). No differences were found in the RNA of cytokines. An association between cold ischemia and chronic rejection was observed in experimental vascularized composite allotransplantation. Chronic rejection intensity and distal progression were significantly related with cold ischemia. The leukocyte infiltrates in vascularized composite allotransplantation components were a rejection marker; however, their exact implication in monitoring and their relation with cold ischemia are yet to be clarified.

Near Normothermic Ex Situ Perfusion of Human Limbs for 24 Hours: N/A - Not a clinical study

There is no financial information to disclose. Currently vascularized composite allografts (VCA) are cold preserved (4°C) until transplantation.1Hartzell T.L. Benhaim P. Imbriglia J.E. Shores J.T. Goitz R.J. Balk M. et al.Surgical and technical aspects of hand transplantation: is it just another replant?.Hand Clin. 2011; 27: 521-530Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar This process is time limited, as the tissue has to be revascularized within 4-6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation.2Rojas-Pena A. Reoma J.L. Krause E. Boothman E.L. Padiyar N.P. Cook K.E. et al.Extracorporeal support: improves donor renal graft function after cardiac death.Am J Transpl. 2010; 10: 1365e-1374eCrossref PubMed Scopus (33) Google Scholar This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury.3Murata S. Miniati D.N. Kown M.H. Koransky M.L. Lijkwan M.A. Balsam L.B. et al.Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts.Transplantation. 2004; 78: 1166-1171Crossref PubMed Scopus (27) Google Scholar Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours.4Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. Ex-situ limb perfusion system to extend vascularized composite tissue allograft survival.Transplantation. 2015; 99: 2095-2101Crossref PubMed Scopus (37) Google Scholar, 5Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. The effect of ex situ perfusion in a swine limb vascularized composite tissue allograft on survival up to 24 hours.J Hand Surg Am. 2016; 41: 3-12Abstract Full Text Full Text PDF Scopus (45) Google Scholar In this study, we aimed to test this system on human limb allografts. Four human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature controlled (30-33°C) ex situ perfusion system (Figure) for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4-6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 98±2 mmHg, perfusion flow 319±18 mL/min (∼6-8% of the donor’s estimated cardiac output), and vascular resistance 158±18 mmHg/mL/min. Perfusate had an average pH of 7.43±0.04, pCO2 32±1 mmHg, pO2 317±18 mmHg, and Hb 4.8±0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment and H& E staining showed mild to moderate fatty infiltration on some myocytes at 24 hours. •All limbs appeared viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation.•While no conclusions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area.•It also has the potential to circumvent complications associated with cold preservation.

Contrastive study on cryopreservation of rat amputated limb with UW solution and HTK solution

Objective To compare the effect on preserving rat amputated extremities during the cold preservation between the UW solution and HTK solution. Methods Thirty healthy adult Wistar rats were randomly divided into 3 groups (10 rats in each group) for producing the models of amputated limbs of rats. The three groups were separately irrigated the amputated limbs with UW solution, HTK solution and saline from femoral artery. After irrigation, the extremities were preserved in the refrigerator at 4 ℃. Samples of skeleton muscle tissue were taken for pathological and biochemical examination every 6 hours since the amputation. Results After perfusion, HE: changes of organizational structures of skeletal muscle were lighter in experimental groups than in control group (saline-perfusion group) at the same time. While samples in UW solution-perfusion group has lighter changes of organizational structure than those in HTK solution-perfusion group. TEM: at 12 h, all the three groups have shown the mitochondria edema. At 24 h, the myofibril in saline-perfusion group become fragments. Biochemical examination: at 24 h, indexes in saline-perfusion group, HTK solution group and UW solution group were: ALP (U/L) 3.62 ± 1.32, 3.37 ± 0.84 and 2.68 ± 1.59, respectively; ALT (U/L) 542.25 ± 129.36, 521.82 ± 97.32 and 462.53 ± 74.18, respectively; AST (U/L) 2200.12 ± 687.61, 2002.20 ± 632.27 and 1742.87 ± 396.09, respectively; CK(U/L) 313190.83 ± 42041.32, 283173.50 ± 31488.87 and 271319.67 ± 41147.52, respectively; LDH(U/L) 32409.50 ± 4253.20, 30382.50 ± 2337.18 and 30047.83 ± 4628.78, respectively; GLU(mmol/L) 0.242 ± 0.041、0.240 ± 0.044 and 0.252 ± 0.049, respectively; LAC (mmol/L) 1.790 ± 0.160, 1.792 ± 0.196 and 1.993 ± 0.366, respectively; SOD(U/mL) 80.82 ± 9.46, 91.62 ± 14.97 and 73.71 ± 10.60, respectively. There were statistical difference among the UW solution-perfusion group and centrol group in ALP and GLU (P < 0.05). UW solution-perfusion group has better results than HTK solution-perfusion group in SOD (P < 0.05). Conclusion Amputated limbs irrigated with organ preservation solution as a way to protect skeletal muscle had great effects on lightening tissue damage and keeping tissue active. Between the two usual organ preservation solution, UW solution had a stronger protective effects on the amputated limbs than HTK solution. Key words: Amputated limbs; Organ preservation solution; Skeletal muscle; Cryopreservation; Rat

Protective role of normothermic machine perfusion during reduced-size liver transplantation in pigs.

The purpose of this study is to explore whether normothermic machine perfusion (NMP) preservation is superior to cold preservation during reduced-size liver transplantation (RSLT) in pigs. Twenty-four healthy Ba-Ma mini pigs were used (aged >13 months; weight 25-35 kg; regardless of sex). The animals were randomized into 2 groups. In group A (NMP), donor livers were harvested without warm ischemia time and heartbeats and then were connected to the NMP system to reduce the livers' size under the normothermic condition. In group B (University of Wisconsin [UW] solution), donor livers were harvested without warm ischemia time and heartbeats after being perfused by UW solution and were then preserved in 0°C-4°C UW solution to reduce the livers' size under cold conditions. After that, liver transplantation without venovenous bypass was performed. General RSLT information of the pigs from the 2 groups was recorded; the serological indices were measured; and routine pathological examination of liver tissue was observed. A significant difference was observed in the intraoperative bleeding between the 2 groups (P < 0.05), whereas no significant difference was found in the other indices (all P > 0.05). Significant differences of alanine aminotransferase levels, aspartate aminotransferase levels, and lactate dehydrogenase levels between the 2 groups were observed between postoperative days 3 and 5 (P < 0.05). Significant differences of lactic acid levels between the 2 groups were observed between postoperative days 2 and 5 (P < 0.05). Compared with the cold preservation group, the liver tissues of the NMP preservation group only rarely experienced liver cell necrosis and maintained integrities in the hepatic sinusoid spaces and endothelial cells. In conclusion, NMP preservation is superior to cold preservation during RSLT in pigs. Liver Transplantation 22 968-978 2016 AASLD.

Concentration of Tumor Necrosis Factor–α and Interleukin-1β in Isolated Porcine Liver Depending on Type of Transgenesis

BackgroundTransgenic animals may serve as organ donors in human organ transplantation. However, the number of the studies addressing all doubts related to this issue is currently insufficient for the clinical application of this approach. The aim of this study was to analyze the hepatic tumor necrosis factor–α (TNF-α) and interleukin-1β (IL-1β) synthesis during a 24-hour cold preservation of the transgenic pig liver, depending on the type of transgenesis. Materials and MethodsThe study was carried out on wild-type and transgenic pig livers with transferred human α1,2-fucosyltransferase (FUT) and/or α-galactosidase (GAL) gene (four groups; n = 6). Harvested livers were perfused for 30 minutes and stored for 24 hours in Biolasol (Biochefa) solution at 4°C with a subsequent 30-minute reperfusion (reflush). TNF-α and IL-1β concentrations were analyzed with an enzyme-linked immunosorbent assay. Perfusates were collected during the initial perfusion as well as after 24 hours of preservation and during the reperfusion. Tissue samples were harvested just after animal sacrifice, and after organ perfusion and reperfusion. ResultsA decrease in TNF-α concentration in homogenates was noted after both perfusion and reperfusion in all experimental groups. In contrast, there was a significant decrease in IL-1β concentration in the group with combined human FUT and GAL transgenes. Concurrently, increases in TNF-α and IL-1β concentrations were observed in the reperfusion perfusates in all groups. ConclusionThis study shows that IL-1β is synthesized in the ischemic livers of the transgenic animals with both human α1,2-fucosyltransferase and α-galactosidase transgenes. Further analysis is required to determine the importance of this observation.

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver.

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48-h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H2 (+) and H2 (-) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H2 (-) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8-hydroxy-2-deoxyguanosine-positive cell rate, apoptotic index, and endothelial endothelin-1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H2 (+) group, these harmful changes were significantly suppressed [vs. H2 (-)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter.

Short Oxygenated Warm Perfusion With Prostaglandin E1 Administration Before Cold Preservation as a Novel Resuscitation Method for Liver Grafts From Donors After Cardiac Death in a Rat In Vivo Model.

We previously demonstrated that short oxygenated warm perfusion (SOWP) prevented warm ischemia-reperfusion injury in rat livers from donors after cardiac death (DCDs) in an ex vivo model. In the present study, we aimed to examine the in vivo effects of SOWP and SOWP with prostaglandin E1 (PGE1) in DCD rat liver transplants. We performed liver transplantation after 6-hour cold preservation using grafts retrieved from DCD rats, divided into nontreatment (NT), SOWP, and SOWP with PGE1 (SOWP + PG) treatment groups. The SOWP grafts were perfused with oxygenated buffer at 37°C for 30 minutes before cold preservation. Prostaglandin E1 was added to the SOWP + PG group perfusate. Eleven liver transplants from each group were performed to evaluate graft function and survival; 5 rats were used for data collection after 1-hour reperfusion, and 6 rats were used for the survival study. As a positive control, the same experiment was performed in a heart-beating donor group. In both the SOWP and SOWP + PG groups, serum liver enzymes, intercellular adhesion molecule 1 levels, and cellular damage were significantly decreased compared with the NT group. In the SOWP + PG group, bile production and energy status were significantly improved compared with the NT group. The 4-week survival was 0% (0/6), 67% (4/6), 83% (5/6), and 100% (6/6) in the NT, SOWP, SOWP + PG, and heart-beating donor group, respectively. Short oxygenated warm perfusion before cold preservation and the addition of PGE1 to SOWP were thus beneficial in an in vivo rat model.

14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2)

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Resveratrol Prevents Warm Ischemia–Reperfusion Injury in Liver Grafts From Non–Heart-Beating Donor Rats

BackgroundSuccessful liver transplantation from non–heart-beating donors (NHBDs) might enlarge donor source. Some studies have reported that resveratrol (RES), an activator of sirtuins, has cytoprotective effects on ischemia-reperfusion (I/R) injury. The purpose of this study was to investigate the effects of RES on warm I/R injury in rats. MethodsMale Wister rats were divided into 5 groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the NHB group, whose livers were retrieved under apnea-induced NHB conditions; (3) the ethanol group, retrieved in the same manner as the NHB group with ethanol (10 μL) as a solvent; (4) the RES-1 group, retrieved in the same manner as the NHB group and pretreated with RES (0.4 mg/kg, dissolved in 10 μL ethanol); and (5) the RES-2 group, retrieved in the same manner as the NHB group and pretreated with RES (2 mg/kg, dissolved in 10 μL ethanol). The resected livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissues were investigated. ResultsThe bile production, portal vein flow volume, tumor necrosis factor-α level, and adenosine triphosphate level in the RES-2 group were significantly improved compared with in the NHB group. Histology revealed numerous well-preserved sinusoidal endothelial cells in the RES-2 group. ConclusionsRES might reduce warm I/R injury and improve the viability of liver grafts from NHBDs. We considered that this method may represent a promising approach for clinical liver transplantation from NHBDs.

Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.

Attenuation of Pulmonary Ischemia-Reperfusion Injury by Adenosine A2B Receptor Antagonism

Ischemia-reperfusion injury (IRI) is a major source of morbidity and mortality after lung transplantation. We previously demonstrated a proinflammatory role of adenosine A2B receptor (A2BR) in lung IR injury. The current study tests the hypothesis that A2BR antagonism is protective of ischemic lungs after invivo reperfusion or exvivo lung perfusion (EVLP). Mice underwent lung IR with or without administration of ATL802, a selective A2BR antagonist. A murine model of EVLP was also used to evaluate rehabilitation of donation after circulatory death (DCD) lungs. DCD lungs underwent ischemia, cold preservation, and EVLP with Steen solution with or without ATL802. A549 human type 2 alveolar epithelial cells were exposed to hypoxia-reoxygenation (HR) (3 hours/1 hour) with or without ATL802 treatment. Cytokines were measured in bronchoalveolar lavage (BAL) fluid and culture media by enzyme-linked immunoassay (ELISA). After IR, ATL802 treatment significantly improved lung function (increased pulmonary compliance and reduced airway resistance and pulmonary arterypressure) and significantly attenuated proinflammatory cytokine production, neutrophil infiltration, vascular permeability, and edema. ATL802 also significantly improved the function of DCD lungs after EVLP(increased compliance and reduced pulmonary artery pressure). After HR, A549 cells exhibited robust production of interleukin (IL)-8, a potent neutrophil chemokine, which was significantly attenuated by ATL802. These results demonstrate that A2BR antagonism attenuates lung IRI and augments reconditioning of DCD lungs by EVLP. The protective effects of ATL802 may involve targeting A2BRs onalveolar epithelial cells to prevent IL-8 production. A2BR may be a novel therapeutic target for mitigating IRI to increase the success of lung transplantation.

(472) - The Interleukin 4 Receptor-α Potentiates the Effect of Oncostatin M Receptor on the Release of FGF23 by Cardiomyocytes

are combined, there are concerns regarding delays to institution of liver cold preservation. The aim of this study was to develop a combined DCD heart and liver retrieval protocol in a pig model with subsequent normothermic machine perfusion (NMP) of both organs to mitigate warm ischemic damage. Methods: Pigs (n= 12; 60-70 kg) were anesthetised. Baseline observations were recorded. After sternotomy, and laparotomy, ventilatory support was withdrawn to mimic DCD conditions. Death was defined as equalisation of central venous and mean arterial pressures. After a 5-minute standoff period, 1.5-2 L blood was collected from a right atrial cannula then a cardiac preservation flush (4°C) commenced via the proximal ascending aorta. The inferior vena cava was cut to vent the organs. The thoracic cavity was kept cold with saline ice slush. The heart was explanted and prepared on back-table for NMP. Liver preservation was begun immediately after blood collection by cold preservation solution flush via the infra-renal aorta and portal vein. The hepatic artery, portal vein and common bile duct were transected, and the liver explanted for back-table NMP preparation. Results: Warm ischaemic time (WIT) and back-table time (BTT) was 21 + 5 and 29 + 6 min for the heart; 20 + 6 (commencement of flush) and 33 + 19 min for the liver. The average blood volume collected was 1.6 L: 1:1 dilution with Krebs resulted in significant hemodilution (from 23 + 5% to 15 + 6%) and hypocalcemia (1.30 + 0.15 to 0.64 + 0.32 mM). This resulted in sub-optimal cardiac contractile recovery despite a favourable lactate profile. Liver enzyme release, bile production, and lactate and pH profiles were favourable during the first 4-6 hours of NMP but deteriorated thereafter. Conclusion: Heart and liver retrieval under standard DCD protocol is possible without excessively extending the WIT for either organ. However, there is insufficient donor blood to support NMP of both organs.