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Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37◦C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4◦C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10◦ or 15◦C. At 21◦C some re-fusion was visible, which became more marked at 25◦C. Networks of tubular mitochondria similar to control cells only re-appeared at 37◦C. ATP content decreased at 4◦C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/106 cells and decreased even further when rewarming cells to 10◦ and 15◦C. Values after rewarming at 21◦C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4◦C incubation and recovered with increasing temperatures to 36 ± 10% at 25◦C and 78 ± 17% at 37◦C. Integrity of monolayers, largely disturbed at 4◦C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15◦C upwards, complete recovery at 37◦C. Endothelial repair processes (scratch assay) at 25◦C were clearly inferior to those at 37◦C. These data suggest that reconditioning temperatures below 21◦C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25◦C appear required, with 30◦C being superior and 37◦C yielding the best results.

Organ shortage and the increasing use of extended criteria donor grafts for transplantation drives efforts for more efficient organ preservation strategies from simple cold storage toward dynamic organ reconditioning. The choice of a suitable preservation solution is of high relevance in different organ preservation or reconditioning situations. Custodiol-MP is a new machine perfusion solution giving the opportunity to add colloids according to organ requirements. The present study aimed to compare new Custodiol-MP with clinically established Belzer MPS solution. Porcine kidneys were ischemically predamaged and cold stored for 20 hours. Ex vivo machine reconditioning was performed either with Custodiol-MP (n = 6) or with Belzer MPS solution (n = 6) for 90 minutes with controlled oxygenated rewarming up to 20°C. Kidney function was evaluated using an established ex vivo reperfusion model. In this experimental setting, differences between both types of perfusion solutions could not be observed. Machine perfusion with Custodiol-MP resulted in higher creatinine clearance (7.4 ± 8.6 mL/min vs. 2.8 ± 2.5 mL/min) and less TNC perfusate levels (0.22 ± 0.25 ng/mL vs. 0.09 ± 0.08 ng/mL), although differences did not reach significance. For short-term kidney perfusion Custodiol-MP is safe and applicable. Particularly, the unique feature of flexible colloid supplementation makes the solution attractive in specific experimental and clinical settings.

Oximes play an essential role in the therapy of organophosphorus compound (OP) poisoning by reactivating inhibited acetylcholinesterase. Impairment of liver function was observed in OP poisoning and associated with obidoxime treatment by some reports. In this study human three-dimensional HepaRG spheroids were used as complex in vitro model to investigate oxime-induced liver toxicity. In this context, cold storage of liver spheroids at 4 °C in standard culture medium and in optimized tissue preservation solutions of up to 72 h was assessed. Cold storage in standard culture medium resulted in a complete loss of viability whereas an optimized tissue preservation solution preserved viability. Separately from that liver spheroids were exposed to the four oximes pralidoxime, obidoxime, HI-6, MMB-4 and cytotoxicity (effective concentration, EC₅₀) was determined with an ATP-based assay at several time points. The release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin secretion was measured in supernatants. The same parameters were assessed with diclofenac as positive hepatotoxic control and with the OP pesticides malathion and malaoxon alone or in the presence of obidoxime. All individual tested oximes and OP showed a low cytotoxicity with effective concentrations mostly >2,000 μM. In contrast, the exposure to malaoxon in the presence of 1,000 μM obidoxime resulted in a marked decrease of viability and an increased release of AST indicating risk of liver injury only if oxime antidotes are strongly overdosed.

BACKGROUND HTK-N was developed based on the traditional HTK preservation solution, resulting in stronger protection against reactive oxygen species as well as better tolerance to hypothermia and ischemia. Aim of the present study was to compare HTK-N to HTK in clinical kidney transplantation demonstrating safety and non-inferiority. METHODS We performed a randomized controlled single blinded clinical phase II trial in patients undergoing living donor kidney transplantation. After retroperitoneoscopic nephrectomy kidneys were either perfused and stored with classical HTK solution or the new HTK-N solution. Primary endpoint was the glomerular filtration rate (eGFR according to CKD EPI) 3 months after transplantation. Secondary endpoints included graft and patient survival beside others. RESULTS The study included 42 patients, of which 22 were randomized in the HTK-N group and 20 in the HTK group. The primary end point showed a mean eGFR of 55.4 ± 14.0 ml/min/1.73m2 in the HTK group compared to a GFR of 57.2 ± 16.7 ml/min/m2 in the HTK-N group (p = 0.72). Regarding secondary endpoints, there were no apparent differences. Posttransplant graft and patient survival was 100%. CONCLUSION This study is the first clinical application of HTK-N for kidney preservation and demonstrates non-inferiority compared to HTK in the setting of living donor kidney transplantation. This article is protected by copyright. All rights reserved.

Purpose Ex vivo lung perfusion is an innovative technique to evaluate marginal lung organs especially after DCD. Normothermic continuous perfusion should reduce ischemic damage and improve the outcome of lung transplantation. However, the optimal protocol for EVLP has not been defined so far. The aim of our study was to compare cytokine/chemokine concentrations in perfusion solutions using different kinetics and solution compounds of EVLP in a porcine cardiac arrest model and to correlate the inflammatory parameters to oxygenation capacity values (∆pO2). Methods Following cardiac arrest and 1h of warm ischemia, lungs were harvested and flushed. Groups were processed as immediate (I-EVLP) 1h cold static preservation (CSP) and delayed (D-EVLP; 9h CSP). D-EVLP lungs were perfused with different solutions: Steen vs. modified Custodiol-N containing dextran (CD) or dextran/albumin (CDA). Cytokine/chemokine levels were analyzed at baseline (0h), after 1h, 4h using multiplex protein arrays. Results Concentrations of TNF-α, IL-6, CXCL8, IFN-γ, IL-1α and IL-1β increased significantly (p Conclusion In a non-heart beating porcine cardiac arrest model with relevant IRI, lungs were perfused with normothermic EVLP. Longer CSP prior to EVLP did not result in enhanced cytokine secretion, but the first hours of reperfusion seem crucial for tissue damage. CD-solution dampens the cytokine/chemokine secretion probably by iron chelators and, possibly, protecting effects of dextran. Addition of albumin had no further effect on inflammation. Cytokine/chemokine concentrations correlated negatively with the oxygenation capacity, an important parameter for organ acceptance. These finding may help to optimize the ex vivo preservation procedure and possibly, more organs could reach the clinically relevant threshold for transplantation, thus, the pool of marginal donor lungs could be enlarged.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

The use of primary hepatocytes in pharmacological and toxicological research as well as for clinical and biotechnological applications requires adequate storage options for these cells. However, hepatocytes are very susceptible to cryopreservation injury. Based on experience in hypothermic storage of hepatocytes, we, in this study, aimed to optimize hepatocyte cryopreservation.Rat hepatocytes were cryopreserved in serum-containing cell culture medium or in serum-free solutions optimized for hypothermic storage, all with 10% dimethyl sulfoxide, using a standard protocol (-1°C/min in a controlled-rate freezer). After rapid thawing, cells were seeded in supplemented Leibovitz-15 cell culture medium without further purification steps. Cell attachment and metabolic activity were assessed.Cell attachment (37% ± 15% vs. 9% ± 7% of noncryopreserved control cells) and metabolic activity (resazurin reduction: 47% ± 23% vs. 25% ± 8%; glucose release: 44% ± 6% vs. 15% ± 7%; and urea production: 31% ± 16% vs. 5% ± 8%) were significantly higher after cryopreservation in the new solution compared to cryopreservation in cell culture medium. Experiments with modified solutions suggested that the protective effect of the new solution is multifactorial.In summary, significant improvement of cell attachment and function compared to cell culture medium was achieved after cryopreservation in serum-free hepatocyte cold storage solution.

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers. The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at −80°C, monolayers were rapidly thawed and re-cultured in cell culture medium. Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of −1°C/min. In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.

Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

Purpose Ex Vivo Lung Perfusion (EVLP) is a promising tool for evaluation and reconditioning in marginal donor lungs. However, the optimal protocol is discussed controversially. It is our hypothesis that different EVLP protocols and different perfusion solutions result in different cytokine patterns during EVLP. Methods Following cardiac arrest and 30 min of no-touch warm ischemia, pig lungs were harvested, flushed and stored in cold preservation solutions. Different groups were processed as immediate long EVLP (I-EVLP, 12 h), delayed short EVLP (4 h) after 9 hours of cold storage (D-EVLP). Lungs were perfused with different acellular solutions: Steen solution or modified HTK solution containing either dextran(CD) or dextran and albumin (CDA). Pulmonary gas exchange, biochemical and structural parameters were recorded hourly. Cytokine / chemokine levels were analyzed in perfusates at baseline and after 1 and 4 h of EVLP. Results ∆ pO2 (pulmonary venous - pulmonary arterialpO2) was higher in D-EVLP group, especially at h 1: I-EVLP: 259 ± 132, D-EVLP: 291 ± 96 and h 2: I-EVLP: 228 ± 125, D-EVLP: 252 ± 116 (n.s.). Compared to D-EVLP, lungs that underwent EVLP with CD and CDA showed significantly higher oxygen capacity (∆ pO2 CD: 402.79 +30.33 mmHg, CDA: 414.86 + 9.77 mmHg). Within 4 h ELVP, concentrations of IFN-γ, TNF-a, IL-1α/β, IL-10, CXCL8 (IL-8) and IL-18 increased significantly (all p Conclusion In a non-heart beating porcine lung donor model with relevant ischemic tissue damage, lungs were perfused with normothermic EVLP. Even after 9 hours cold static preservation (CSP) and 4 hours EVLP lung, function is comparable to direct EVLP. The modified HTK solution with dextran and dextran/ albumin had significantly better oxygenation. Cytokines and chemokines including the cell death marker IL-18 increase during 4 h of EVLP but a delay before EVLP may not dramatically worsen the inflammatory response.

Background The small bowel is prone to ischemic injury during transport before transplantation, an injury that endangers the recipient patient. The small-bowel mucosal microcirculation in particular appears to be highly sensitive to injury. Current preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution provide some protection to the graft. However, these were developed decades ago and do not address several critical processes, such as hypoxia-induced membrane pores and free radical-mediated hypothermic injury. Methods To protect the graft from cold ischemic injury, we implemented a modified HTK solution here, including glycine, alanine, and iron chelators in a heterotopic, syngeneic small-bowel transplantation model of the rat. The effects of the modified solution and its major components were compared against the conventional HTK solution using intravital microscopy in the early reperfusion period. Results The amino acid glycine, added to HTK solution, slightly improved mucosal perfusion. Both, the modified base solution (without iron chelators) and iron chelators increased functional capillary density of the mucosa during the early reperfusion period. The complete modified solution (with glycine, alanine, and iron chelators) significantly increased the perfusion index, functional capillary density of the mucosa, and red blood cell velocity in the grafts after reperfusion in comparison with the grafts preserved with HTK. Conclusions The modified preservation solution improved the microcirculation of the transplants and needs detailed evaluation in further models of small-bowel transplantation.

Summary Custodiol-N, a new preservation solution, has been shown particularly suitable for hypothermic machine perfusion preservation (HMP) in isolated porcine kidneys. These preliminary results should be confirmed in an actual transplant model in vivo. Kidney function after 21 h of HMP was studied in an autotransplant model using Landrace pigs (25–30 kg; n = 6 per group). Perfusion was performed with oxygenated perfusate, using either Custodiol-N solution including 50 g/l dextran 40 (CND) or kidney perfusion solution 1 (KPS-1) as gold standard. Viability of the grafts was followed for 1 week after bilateral nephrectomy in the recipient pigs. HMP with CND resulted in less acute tubular injury, evaluated by levels of fatty acid-binding protein and better clearance function during the first 24 h after Tx than with KPS-1 (P

Background Hypothermic machine perfusion (HMP) for preservation led to compelling success for outcomes of renal transplantation. Application of different concentrations of oxygen during renal HMP has not been systematically analyzed. This study investigates the aspects of renal function and morphology in dependence of oxygen concentrations during HMP in a porcine donation after circulatory death model. Methods After 30 min of warm ischemia, porcine kidneys were randomly assigned to preservation for 21 hr by HMP without oxygenation (HMPnoox), oxygenated HMP with air (HMPair), or 100% oxygen (HMPox100%). Afterward, kidneys were reperfused for 2 hr in an ex vivo model for assessment of function and integrity. Results Application of HMPox100% led to significantly increased blood flow during reperfusion compared to HMPnoox. Preservation by HMPox100% led to a doubling of creatinine clearance after 90 and 120 min of reperfusion (13.4 and 12.0 mL/min) compared to preservation by HMPnoox (7.3 and 7.7 mL/min; P=0.01). Oxygenated HMP with air led to results between the two other groups. Fractional excretion of sodium demonstrated a strong tendency of higher values after HMPnoox compared to HMPox100% (P=0.096) and HMPair (P=0.09). Analysis of structural integrity during reperfusion demonstrated significantly higher values of lactate dehydrogenase resembling cell damage (P=0.02), higher values of gamma-glutamyl-transferase (gGT) resembling tubulus injury (P=0.048), and more pronounced tubular dilatation (P=0.02) after HMPnoox compared to HMPox100%. Conclusion The present study demonstrates that application of HMPox100% for kidneys from donations after circulatory death results in better renal function during early reperfusion compared to HMPnoox.

Organ shortage leads to an increased utilization of marginal organs which are particularly sensitive to storage-associated damage. Cold incubation and rewarming-induced injury is iron-dependent in many cell types. In addition, a chloride-dependent component of injury has been described. This work examines the injury induced by cold incubation and rewarming in isolated rat renal proximal tubules. The tissue storage solution TiProtec® and a chloride-poor modification, each with and without iron chelators, were used for cold incubation. Incubation was performed 4C for up to 168 h, followed by rewarming in an extracellular buffer (3 h at 37C). After 48, 120 and 168 h of cold incubation LDH release was lower in solutions containing iron chelators. After rewarming, injury increased especially after cold incubation in chelator-free solutions. Without addition of iron chelators LDH release showed a tendency to be higher in chloride-poor solutions. Following rewarming after 48 h of cold incubation lipid peroxidation was significantly decreased and metabolic activity was tenden-tially better in tubules incubated with iron chelators. Morphological alterations included mitochondrial swelling and fragmentation being partially reversible during rewarming. ATP content was better preserved in chloride-rich solutions. During rewarming, there was a further decline of ATP content in the so far best conditions and minor alterations under the other conditions, while oxygen consumption was not significantly different compared to non-stored control tubules. Results show an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and reveal a benefit of chloride for the maintenance of tubular energy state during cold incubation. OA gold

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research.

Abrupt temperature shift from hypothermia to normothermia incurred on reperfusion of organ grafts has been delineated as a genuine factor contributing to reperfusion injury and graft dysfunction after transplantation.In a first clinical series of 6 patients, cold-stored livers, all allocated by the rescue offer mechanism by Eurotransplant, were subjected to machine-assisted slow controlled oxygenated rewarming (COR) for 90 minutes before engrafting. A historical cohort of 106 patients basically similar in graft (all rescue offer organs) and recipient factors was used for comparison.The clinical benefit of COR was documented by a significant reduction by approximately 50% in peak serum transaminases after transplantation compared to untreated controls (AST 563.5 vs. 1204 U/L, P = 0.023). After 6 months graft survival was 100% in the COR group and 80.9% in the controls (P = 0.24). Respective patient survival was 100% and 84.7% (P = 0.28). Real-time assessment of glucose concentration in the perfusion solution correlated well with postoperative synthetic graft function (r = 0.78; P0.02). All treated recipients had normal liver function after a 6-month follow-up and are well and alive.This first clinical application suggests that controlled graft rewarming after cold storage is a feasible and safe method in clinical praxis and might become an adjunct in organ preservation.

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0-4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.The re-beating time was significantly shorter in hearts stored in the new solution (10.3±2.6 min vs. HTK 14.2±4.1 min; p0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3±0.4 vs. HTK 1.6±0.5; p0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8±0.2 vs. HTK 2.2±0.7), less interstitial fibrosis (4.8±1.9% vs. HTK 8.5±3.8%, p0.05), less vasculopathy (14.7±6.9% vs. 22.0±23.2%; p=0.06) and lower TGF-β1-expression (6.6±1.4% vs. HTK 12.0±4.6%).The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-L-histidine, glycine, alanine, arginine and sucrose.

Recent in vitro studies of cold-induced cell injury have revealed the detrimental effects of extracellular chloride on cold-stored isolated rat hepatocytes; however, its influence on endothelial cells is beneficial. To determine which of these effects is predominant in vivo, we tested both a chloride-poor variant of a new histidine-tryptophan-ketoglutarate (HTK)–based preservation solution and a chloride-containing variant in a rat liver transplantation model. The study, which was carried out in a blinded fashion with 7 or 8 rats per group, was divided into 2 parts: (1) a comparison of survival in 3 series under different conditions [different microsurgeons, rat strains, cold ischemia times (3, 12, and 24 hours), and warm ischemia times] and (2) an assessment of the microcirculation (30-90 minutes after reperfusion), laboratory data, bile production, and histology. In each of the survival experiments, a (strong) tendency toward prolonged survival was observed with the new chloride-containing solution (50% versus 12.5%, 75% versus 37.5%, and 100% versus 71.4% [chloride-containing vs. chloride-poor], overall P < 0.05). Additionally, the sinusoidal perfusion rates (83.9% ± 4.0% versus 69.2% ± 10.8%, P < 0.01) and the red blood cell velocities in sinusoids (147.7 ± 26.7 versus 115.5 ± 26.0 μm/second, P < 0.05) and in postsinusoidal venules (332.4 ± 87.3 versus 205.5 ± 53.5 μm/second, P < 0.01) were clearly higher with chloride. Moreover, the serum activities of liver enzymes were slightly reduced (not significantly), and bile production was significantly increased. These results suggest an overall beneficial effect of chloride in HTK-based liver preservation solutions. Liver Transpl 17:650-660, 2011. © 2011 AASLD.

Background The present study was undertaken to investigate the putative benefit of a new, modified HTK solution for hypothermic machine perfusion using a model of rat livers from non-heart beating donors. Methods Livers were retrieved 30 min after cardiac arrest of male Wistar rats and put on a recirculating machine perfusion device. Hypothermic machine perfusion (HMP) was performed for 18 h at 4°C and a rate of 0.5 mL/g−1 min−1 while the preservation solution was oxygenated with 100% O2. Each liver was randomly assigned to the use of one of the following preservation solutions: HTK (histidine-tryptophan-ketoglutarate solution); Custodiol-N base solution (modified HTK-solution) without additives or with the addition of 25 μM deferoxamine + 2.5 μM (Custodiol-N, 2.5) or 7.5 μM (Custodiol-N) of the permeable iron chelator LK 614. Viability of livers was evaluated upon reperfusion in vitro with Krebs-Henseleit buffer according to previously validated techniques for 120 min at 37°C. Results The use of Custodiol-N base solution led to a significantly decreased release of ALT or LDH during HMP and after reperfusion, which was further reduced by Custodiol-N and minimal use of Custodiol-N, 2.5. Only the use of Custodiol-N, 2.5 resulted in an improvement of metabolic activity upon reperfusion, as evaluated by hepatic production of C02, significantly reduced cleavage of caspase 9, and abrogated positive signs of cellular of apoptosis [terminal deoxynucleotide transferase-mediated deoxy-UTP nick-end labeling (TUNEL)-test)]. Conclusion The data presented provide first experimental evidence for enhanced organ protective potential of the new Custodiol-N solution compared with HTK upon hypothemic machine preservation of marginal liver grafts. Moreover, for continuous perfusion preservation the dosage of the lipophilic chelator LK 614 should probably be lower than for static cold storage.

In previous studies we reported on the catalase-like activity and antioxidative properties of a non-heme Fe(III)-tetraaza[14]annulene complex, 5,4-didehydro-5,9,14,18-tetraaza-di(2,2-dimethyl-[5,6]benzo[1,3]dioxolo)[a,h]cyclotetradecene--Fe(III) chloride (TAA-1/Fe). We proposed that intracellular application of the parent, iron-free tetraaza[14]annulene ligand, TAA-1, as precursor would allow antioxidative defense along two lines, i.e. by chelation of potentially toxic cellular iron ions and, subsequently, by catalase-mimic activity. We here set out to establish whether the active catalase mimic is indeed formed intracellularly when cells are loaded with the ligand. When isolated rat hepatocytes were preloaded with TAA-1, they were protected against iron-induced cell injury and oxidative stress elicited by exposure to the membrane-permeable iron complex Fe(III)/8-hydroxyquinoline. After lysis of the cells, followed by ultrafiltration to remove endogenous catalase, the lysate exhibited catalase-like activity, while lysates of control cells not treated with TAA-1 showed no catalase-like activity. By comparison with authentic TAA-1/Fe, an intracellular formation of 2.0 +/- 0.3 microm of the active catalase mimic in native hepatocytes exposed to TAA-1 and of 6.5 +/- 1.0 microm in hepatocytes exposed to both TAA-1 and iron ions was estimated. The intracellular formation of the active catalase mimic thus renders TAA-1 an attractive compound for protection against iron- and/or hydrogen peroxide-dependent cell injuries.

The aim of the present study was to evaluate the potential benefit of two different techniques for the provision of tissue aerobiosis upon cold preservation of marginal livers from non-heart beating donors using a recently developed improved preservation solution. Rat livers were harvested 30 min after cardiac arrest, flushed via the portal vein and cold-stored in HTK or modified HTK-solution (Custodiol-N) for 18 h at 4 degrees C. Other organs were flushed with Custodiol-N and subjected to aerobic conditions by either vascular systemic oxygen persufflation (VSOP) of the cold stored organ or hypothermic machine perfusion (HMP) with oxygenated Custodiol-N. Viability of the livers was assessed after 18 h of preservation by warm reperfusion in vitro for 120 min. Free radical mediated lipid peroxidation was significantly abrogated by the use of Custodiol-N in all groups compared with HTK. Custodiol-N improved enzyme leakage upon reperfusion and histological integrity, but had no impact on functional recovery (bile production, energetic status). However, VSOP further minimized enzyme release during the whole reperfusion period, led to a rise in hepatic bile production and enhanced recovery of energy charge (p0.05, resp. vs Custodiol-N). Histological appearance was concordantly improved in VSOP. During the first 45min of reperfusion, leakage of ALT and LDH was also reduced by MP but deteriorated thereafter and became significantly higher compared to Custodiol-N at the end of the experiment. In conclusion, the results of the present study recommend the use of gaseous oxygen persufflation to improve tissue integrity and functional recovery of predamaged livers.

BACKGROUND Inadequate liver regeneration is still an unsolved problem in major liver resection and living donor liver transplantation (LDLT). Therefore, we have investigated the use of erythropoietin (EPO) as an exogenous stimulator of liver regeneration in rat models of liver resection and LDLT. METHODS Rats were treated with EPO or heat-inactivated EPO-vehicles. Animals underwent 70% or 90% partial hepatectomy (PH) or 30% partial liver transplantation (pLTx). Serum and liver samples were taken to investigate liver function, liver-to-body weight ratio (LBWR), hepatocyte-proliferation (Ki-67), apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-assay), proregenerative cytokines (interleukin [IL]-6/tumor necrosis factor-alpha), and angiogenesis. Gene expression was assessed by in-house cDNA array and quantitative real-time polymerase chain reaction. As clinical parameters, LBWR and overall survival were determined. RESULTS Erythropoietin led to improved liver regeneration as shown by an increased LBWR/Ki-67 after PH and pLTx. Liver damage, indicated by the serum activity of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase was reduced after PH. After surgery EPO treatment induced modulation of c-jun, IL-6, p53, and the antiapoptotic gene Bcl-XL, which was accompanied by a decreased apoptosis rate (0.56% vs. 1.03%; P

We have previously shown that cold-induced injury to hepatocytes and liver endothelial cells occurs predominantly via an iron-dependent pathway. However, other groups have reported evidences suggesting that Ca(2+) ions could be involved in the process of cold-induced injury of liver cells. We here assessed the relative importance and potential interaction of both pathways in cultured primary hepatocytes and cultured liver endothelial cells. The sequence cold incubation/rewarming of hepatocytes and endothelial cells led to an increase in the cytosolic calcium concentration during the early rewarming phase, but the increased cytosolic calcium concentration did not correlate with cell injury. A partial protection from cold-induced cell injury was achieved by the intracellular calcium chelators Quin-2 and BAPTA. However, additional experiments showed that the ability of these chelators to bind iron was probably responsible for a major part of this protection. Incubation in calcium-free media led to an increased cell injury and a physiological calcium concentration (2.5mM) was protective. In addition, targeting suggested downstream pathways of calcium-dependent cold-induced injury, i.e. by the addition of Ruthenium Red, an inhibitor of mitochondrial Ca(2+) uniporter, or by inhibiting Bax translocation to the mitochondria, did not provide protection from cold-induced injury in both cell types. Taken together, our data suggest that calcium increases but does not play a major role in cold-induced cell injury to hepatocytes and liver endothelial cells.

HISTORY AND CLINICAL FINDINGS An 18-year-old patient had for 6 days been suffering from right upper abdominal pain, weight loss, vomiting and yellow discoloration of the skin. For the preceding 8 months he had been regularly taking 1-2 tablets of "ecstasy" (3,4-methylenedioxymethamphetamine--MDMA) per week, the last 8 days before the onset of the described signs. Physical examination was unremarkable, except for pain on pressure over the right upper abdomen and the jaundice. INVESTIGATIONS The activities of SGOT (756 U/I), SGPT (1450 U/I). gamma GT (164 U/I) and lactate dehydrogenase (539 U/I) as well as total bilirubin level (7.5 mg/dl) were elevated. The synthesising functions of the liver were impaired (thromboplastin time 47%, fibrinogen 116 mg/dl). Abdominal sonography was unremarkable. All virological tests (hepatitis A, B, C and D; Epstein-Barr virus; cytomegalovirus; HIV 1 and 2) were negative. TREATMENT AND COURSE The suspected diagnosis was acute liver failure after "ecstasy" intake. The cholestasis and the parameters of liver synthesis and hepatocellular functions deteriorated under symptomatic treatment. 15 days after onset of the first symptoms progressive hepatic encephalopathy occurred and required heterotopic auxiliary liver transplantation (piggy-back technique). 5 months later hepatobiliary sequential scintigraphy demonstrated regenerating of the patient's own liver an atrophy of the transplanted liver. Immunosuppression with cyclosporin A and prednisolone was gradually reduced, and the transplant was removed 6 months postoperatively because of an abscess in it. 11 months after the transplantation liver functions is normal and the patient well. CONCLUSION In young patients with jaundice of unknown origin toxic hepatitis after "ecstasy" intake should be considered. Auxiliary liver transplantation can lead to regeneration during temporary relief of the patient's own liver. After its function has been restored immunosuppression is no longer needed.

Background To improve the rate of successful heart transplantations, organ preservation should be optimized in cardiac transplantation surgery. Iron-dependent oxidative damage and iron-independent, chloride-dependent injury of the endothelium have been described after cold ischemic storage and reperfusion, leading to an enhanced rate of complications and unfavorable outcomes. This screening study tested the effects of iron chelator supplementation in different histidine–tryptophan–ketoglutarate (HTK) organ preservation solutions on endothelial function in a long-term storage model of the isolated rat aorta. Methods Isolated rat aortic rings underwent a 24-hour cold ischemic preservation in different HTK solutions supplemented with iron chelators of low (deferoxamine) and high (LK-614) membrane permeability. In vascular reactivity measurements we investigated the phenyleprine-induced contraction and both endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside with and without an additional external oxidant injury during re-oxygenation. Results Traditional HTK solution, Custodiol, failed to prevent endothelial dysfunction in our experiments. Use of chloride-poor HTK solutions containing N-α-acetyl- l -histidine with and without supplementation with LK-614, but not with deferoxamine, resulted in significant improvement of impaired endothelial function; moreover, complete protection of the endothelium was feasible after 24-hour cold storage. Endothelium-independent functions of vascular smooth muscle were not affected in any of the groups. Conclusions Our results demonstrate the important pathophysiologic role of iron-dependent oxidative injury in the development of endothelial dysfunction after cold storage, which can be prevented by cell-permeable iron chelators.

Nitric oxide (NO) and hydrogen peroxide (H2O2) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H2O2 (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H2O2 alone. In both cell types, the addition of the NO donor increased H2O2 steady-state levels, although with different kinetics: in hepatocytes, the increase in H2O2 levels was already evident at early time points while in liver endothelial cells it became evident after ≥2 h of incubation. NO exposure inhibited H2O2 degradation, assessed after addition of 50 μM, 200 μM, or 4 mM authentic H2O2, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H2O2 degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NOx). Early inhibition of H2O2 degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H2O2 is due to inhibition of H2O2 degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NOx (prevailing in endothelial cells).

Ischemia-reperfusion injury is a complex phenomenon involving not only intracellular injury processes but also an injurious inflammatory response. Both the intracellular injury processes and the injurious events of the inflammatory response are interconnected in pathogenetic networks. Anoxic cell injury predominates in the ischemic phase. The decreased mitochondrial ATP generation impairs cellular ion homeostasis with activation of hydrolases and loss of selective permeability of cell membranes. Upon resupply of blood, the inflammatory response is initiated. Resident cells of the affected tissue, blood-derived cells, and noncellular elements such as the complement system are activated, and signalling and other molecules are formed at altered rates. Cell injury occurring in the reperfusion phase may either be a consequence of cellular alterations that were already initiated in the ischemic phase or may result from the inflammatory response. The intracellular injurious alterations are in part the same as those involved in anoxic cell injury. In addition, activation of intracellular signalling cascades and of apoptotic pathways may take place. Except for a large decrease in their rates, no significant difference exists between the injury processes during warm and cold ischemia as they become evident during ischemia itself. In contrast, the injury processes of the inflammatory response and of cell injury in the reperfusion phase significantly vary depending on pre-existent warm versus cold ischemia. Because of the netlike characteristics of the pathomechanisms, a multifactorial approach is required to provide protection against ischemia-reperfusion injury.

The synthesis and properties of the new fluorescent nitric oxide cheletropic trap (FNOCT) 14, designed for the trapping and quantification of nitric oxide (NO) production in chemical and biological systems, is described (Scheme 3). The dicarboxylic acid 14 and the corresponding bis[(acetyloxy)methyl] ester derivative 15 of the FNOCT contain a 2-methoxy-substituted phenanthrene group as fluorophoric unit. The fluorescence of the reduced NO adduct of this FNOCT (λexc 320 nm, λem 380 nm) is pH-independent. Trapping experiments were carried out in aqueous buffer solution at pH 7.4 with nitric oxide being added as a bolus as well as being released from the NO donor compound MAHMA NONOate (= (1Z)-1-{methyl[6-(methylammonio)hexyl]amino}diazen-1-ium-1,2-diolate), indicating a trapping efficiency of ca. 50%. In a biological application, nitric oxide was scavenged from a culture of lipopolysaccharide-stimulated rat alveolar macrophages. Under the applied conditions, a production of 11.1 ± 1.5 nmol of NO per hour and per 105 cells was estimated.

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Hydrogen peroxide is involved in many types of cell injury and exerts most of its injurious effects in conjunction with chelatable iron. We previously described a synthetic nonporphyrin iron-containing catalase mimic, TAA-1/Fe. Its ligand TAA-1 was designed for application in biological systems in which it is supposed to fulfill a dual task: it should chelate cellular labile iron and thus form the active catalase mimic, thereby decreasing levels of redox-active iron and enhancing the degradation of hydrogen peroxide. Here, we tested these novel compounds in cellular systems, i.e., in cultured hepatocytes and liver endothelial cells. Both the iron complex, i.e., the complete mimic, and the ligand, i.e., the putative precursor of this mimic, provided protection against endothelial cell injury induced by exogenous hydrogen peroxide. Furthermore, the ligand--but not (or less so) the complex--strongly protected both cell types against iron-dependent hypothermic injury and hepatocytes against iron-induced cell injury and against iron-dependent, histidine-induced injury. Together, these results demonstrate that the putative catalase mimic precursor TAA-1 is able to protect cells against iron- and/or hydrogen peroxide-dependent cell injuries and that--in line with our initial concept--it is likely to exert its protection by both iron chelation and hydrogen peroxide degradation.

Background/Aims We previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool—in the absence of any increase in O 2 − /H 2 O 2 formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis. Methods Cultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise ‘physiological' conditions. Results Incubation with Fe(III)/8-hydroxyquinoline (15 μM/30 μM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis. Conclusions These results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT.

Background. Maintenance of the integrity of the vascular endothelium is a critical issue in liver preservation, but hypothermia, applied for cellular protection, induces apoptotic cell death in liver endothelial cells. This cold-induced apoptosis is mediated by an iron-dependent formation of reactive oxygen species. Here, we study the involvement of mitochondria in this process. Methods. Cultured rat liver endothelial cells were incubated in cold University of Wisconsin solution for 18 hr and subsequently rewarmed in cell culture medium. Mitochondrial morphology and membrane potential were evaluated using laser scanning microscopy. Results. During cold incubation in University of Wisconsin solution, a marked, progressive mitochondrial shortening and a reduction in mitochondrial membrane potential occurred. Rewarming of the cells led to mitochondrial ultracondensation, complete loss of the mitochondrial membrane potential, and subsequent apoptotic cell death. The inhibitors of mitochondrial permeability transition, trifluoperazine and fructose, or iron chelation with deferoxamine did not affect mitochondrial shortening during cold incubation but inhibited ultracondensation, loss of mitochondrial membrane potential, and loss of viability during rewarming. Moreover, in these protected cells, an almost complete reestablishment of the mitochondrial membrane potential and morphology could be observed; the few mitochondria that were irreversibly damaged were incorporated into autophagosomes during cellular recovery. Conclusion. Two apparently independent mitochondrial alterations take place during cold incubation and subsequent rewarming of liver endothelial cells. Cold-induced mitochondrial shortening represents a reversible process, whereas iron-mediated mitochondrial permeability transition and ultracondensation during rewarming are irreversible and constitute an important mediator of cold-induced apoptosis.

Background. We have previously shown that in cultured liver endothelial cells the mere sequence of hypothermia and rewarming induces a pronounced, iron-dependent apoptosis that is likely to contribute to hepatic preservation injury. Here we study the involvement of proteases in this cold-induced apoptosis. Methods. Cultured liver endothelial cells were incubated in preservation solutions at 4°C in the absence or presence of protease inhibitors. Cell injury and different protease activities were assessed. Results. Cold incubation of liver endothelial cells in University of Wisconsin or histidine-tryptophan-ketoglutarate (HTK) solution led to marked cell injury, which was strongly inhibited by the protease inhibitor 3,4-dichloroisocoumarin (DCI) (lactate dehydrogenase release: 86.0±2.6% in HTK and 4.0±0.4% in HTK + DCI after 24 hr of cold incubation). Determination of protease activity showed a doubling in the activity of a Suc-Leu-Leu-Val-Tyr-AMC–cleaving protease and some increase in a relatively low Suc-Ala-Ala-Ala-AMC–cleaving activity after 8 hr of cold incubation in HTK solution or 16 hr in University of Wisconsin solution. Further characterization of the protease activities showed that they belonged to two different proteases, with the major activity being calcium independent, inhibited by DCI, MG-132, and lactacystin, and strongly decreased by immunoprecipitation with an antiproteasome antibody. Preincubation of the cells with the iron chelator deferoxamine prevented the cold-induced increase of both activities. Conclusion. These results show that (i) the proteasome and possibly, in addition, a serine protease are involved in cold-induced apoptosis of liver endothelial cells and (ii) the protease activation is downstream from the increase in intracellular chelatable iron.

Although University of Wisconsin (UW) solution aims at the prevention of cold-induced cell injury, it failed to protect against cold-induced apoptosis of hepatocytes and liver endothelial cells: when incubated in UW solution at 4°C for 24 hours and subsequently rewarmed at 37°C, 72% ± 8% of rat hepatocytes and 81% ± 5% of liver endothelial cells lost viability. In both cell types, the observed cell damage occurred under an apoptotic morphology; it appeared to be mediated by a rapid increase in the cellular chelatable iron pool by a factor ≥2 (as determined in hepatocytes) and subsequent formation of reactive oxygen species (ROS). Consequently, this cell injury was decreased by iron chelators to 6 to 25% (hepatocytes) and 4% ± 2% (liver endothelial cells). Deferoxamine nearly completely inhibited the occurrence of apoptotic morphology in both cell types. In liver endothelial cells, cold-induced apoptosis occurring during rewarming after 24 hours of cold incubation in UW solution was far more pronounced than in cell culture medium (loss of viability: 81% ± 5% vs. 28% ± 13%), but viability could even be maintained for 2 weeks of cold incubation by use of deferoxamine. In conclusion, this pathological mechanism might be an explanation for the strong endothelial cell injury known to occur after cold preservation. With regard to the extent of this iron-mediated injury, addition of a suitable iron chelator to UW solution might markedly improve the outcome of liver preservation.