[Modern concepts for the dynamic preservation of the liver and kidneys in the context of transplantation]. / Moderne Konzepte zur dynamischen Konservierung von Leber und Nieren im Rahmen einer Transplantation.

The increasing demand on donor grafts has forced experimental research on transplantation medicine to develop more efficient organ preservation strategies. Simple cold storage of grafts rarely offers optimal conditions for extended criteria donor organs. Hypothermic, oxygenated machine perfusion (HMP) is a classical method of dynamic organ preservation, which enables the provision of oxygen and nutrients to the tissue and provides a metabolic recovery of the graft prior to implantation. A more modern approach is normothermic machine perfusion (NMP), which instead simulates physiological conditions and enables an ex vivo evaluation and treatment of organ grafts. However, studies have found that a preceding period of cold storage significantly mitigates the functional advantage of NMP. A strategy to circumvent this phenomenon is controlled oxygenated rewarming (COR). The cold-stored graft is slowly and gradually rewarmed to subnormothermic or normothermic temperatures, providing a gentle adaption of energy metabolism and counteracting events of rewarming injury.

Temporal parameters of post-stress prophylactic glucose treatment in rats.

Acute trauma can lead to life-long changes in susceptibility to psychiatric disease, such as post-traumatic stress disorder (PTSD). Rats given free access to a concentrated glucose solution for 24 h beginning immediately after trauma failed to show stress-related pathology in the learned helplessness model of PTSD and comorbid major depression. We assessed effective dosing and temporal constraints of the glucose intervention in three experiments. We exposed 120 male Sprague-Dawley rats to 100, 1 mA, 3-15 s, inescapable and unpredictable electric tail shocks (over a 110-min period) or simple restraint in the learned helplessness procedure. Rats in each stress condition had access to a 40% glucose solution or water. We measured fluid consumption under 18-h free access conditions, or limited access (1, 3, 6, 18 h) beginning immediately after trauma, or 3-h access with delayed availability of the glucose solution (0, 1, 3, 6 h). We hypothesized that longer and earlier access following acute stress would improve shuttle-escape performance. Rats exposed to traumatic shock and given 18-h access to glucose failed to show exaggerated fearfulness and showed normal reactivity to foot shock during testing as compared to their water-treated counterparts. At least 3 h of immediate post-stress access to glucose were necessary to see these improvements in test performance. Moreover, delaying access to glucose for more than 3 h post-trauma yielded no beneficial effects. These data clearly identify limits on the post-stress glucose intervention. In conclusion, glucose should be administered almost immediately and at the highest dose after trauma.

Machine Perfusion of Donor Livers for Transplantation: A Proposal for Standardized Nomenclature and Reporting Guidelines.

With increasing demand for donor organs for transplantation, machine perfusion (MP) promises to be a beneficial alternative preservation method for donor livers, particularly those considered to be of suboptimal quality, also known as extended criteria donor livers. Over the last decade, numerous studies researching MP of donor livers have been published and incredible advances have been made in both experimental and clinical research in this area. With numerous research groups working on MP, various techniques are being explored, often applying different nomenclature. The objective of this review is to catalog the differences observed in the nomenclature used in the current literature to denote various MP techniques and the manner in which methodology is reported. From this analysis, we propose a standardization of nomenclature on liver MP to maximize consistency and to enable reliable comparison and meta-analyses of studies. In addition, we propose a standardized set of guidelines for reporting the methodology of future studies on liver MP that will facilitate comparison as well as clinical implementation of liver MP procedures.

Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion.

The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 20°C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

Hypothermic reconditioning by gaseous oxygen improves survival after liver transplantation in the pig.

The quality of cold-stored livers declines with the extension of ischemic time and the risk of primary dys- or nonfunction increases. Here, we provide in vivo evidence for the efficacy of the previously developed end-ischemic gaseous oxygen persufflation technique to resuscitate liver grafts after extended storage times. Porcine livers were recovered according to standard multiorgan procurement protocol. Control livers were cold stored in histidine tryptophan ketoglutarate solution for 10 h (cold storage [CS]; n = 6) at 4°C. In the treatment group (n = 6), livers were additionally subjected to hypothermic reconditioning (HR) by gaseous oxygen persufflation via the caval vein for 2 h before transplantation. Viability was assessed by orthotopic liver transplantation and 1 week follow-up. HR significantly improved pretransplant energy charge and initial graft function after transplantation. One week survival after CS was 0% whereas five of six pigs (83%) survived in the HR group. At that time, coagulation parameters were in the normal range and histological analysis disclosed healthy liver tissue with normal trabecular architecture in the treated grafts. Molecular analyses identify the prevention of ischemia-induced decline of cellular autophagy and mitigation of innate immune machinery (high-mobility group protein B1, interferon-ß) as operative mechanisms among the protective effects provided by HR.

Meloxicam, a COX-2 inhibitor, ameliorates ischemia/reperfusion injury in non-heart-beating donor livers.

BACKGROUND/AIMS: Chronic organ donor shortage has led to the consideration to expand the donor pool with livers from non-heart-beating donors (NHBD), although a higher risk of graft dys- or nonfunction is associated with these livers. We examined the effects of selective cyclooxygenase-2 (COX-2) inhibition on hepatic warm ischemia (WI) reperfusion (I/R) injury of NHBD. METHODS: Male Wistar rats were used as donors and meloxicam (5 mg/kg body weight) was administered into the preservation solution. Livers were excised after 60 min of WI in situ, flushed and preserved for 24 h at 4°C. Reperfusion was carried out in vitro at a constant flow for 45 min. During reperfusion (5, 15, 30 and 45 min), enzyme release of alanine aminotransferase and glutamate lactate dehydrogenase were measured as well as portal venous pressure, bile production and oxygen consumption. The production of malondialdehyde was quantified and TUNEL staining was performed. Quantitative PCR analyzed COX-2 mRNA. COX-2 immunohistochemistry and TxB(2) detection completed the measurements. RESULTS: Meloxicam treatment led to better functional recovery concerning liver enzyme release, vascular resistance and metabolic activity over time in all animals. Oxidative stress and apoptosis were considerably reduced. CONCLUSION: Cold storage using meloxicam resulted in significantly better integrity and function of livers retrieved from NHBD. Selective COX-2 inhibition is a new therapeutic approach achieving improved preservation of grafts from NHBD.

Rapid avoidance acquisition in Wistar-Kyoto rats.

The relationship between trait stress-sensitivity, avoidance acquisition and perseveration of avoidance was examined using male Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. Behavior in an open field was measured prior to escape/avoidance (E/A) acquisition and extinction. E/A was assessed in a discrete trial lever-press protocol. The signal-shock interval was 60s with subsequent shocks delivered every 3s until a lever-press occurred. A 3-min flashing light safety signal was delivered contingent upon a lever-press (or failure to respond in 5 min). WKY rats displayed phenotypic low open field activity, but were clearly superior to SD rats in E/A performance. As avoidance responses were acquired and reached asymptotic performance, SD rats exhibited "warm up", that is, SD rats rarely made avoidance responses on the initial trial of a session, even though later trials were consistently accompanied with avoidance responses. In contrast, WKY rats did not show the "warm up" pattern and avoided on nearly all trials of a session including the initial trial. In addition to the superior acquisition of E/A, WKY rats demonstrated several other avoidance features that were different from SD rats. Although the rates of nonreinforced intertrial responses (ITRs) were relatively low and selective to the early safety period, WKY displayed more ITRs than SD rats. With removal of the shocks extinction was delayed in WKY rats, likely reflecting their nearly perfect avoidance performance. Even after extensive extinction, first trial avoidance and ITRs were evident in WKY rats. Thus, WKY rats have a unique combination of trait behavioral inhibition (low open field activity and stress sensitivity) and superior avoidance acquisition and response perseveration making this strain a good model to understand anxiety disorders.

Short-term resuscitation of predamaged donor livers by brief machine perfusion: the influence of temperature.

Short-term machine perfusion after liver retrieval from non-heart-beating donors has been considered a beneficial means to reverse deleterious priming of the predamaged organ. In this study, the possible impact of different temperatures during aerobic perfusion was addressed, focusing on liver metabolic functions, structural integrity, and vascular activation. Livers retrieved 30 minutes after cardiac arrest of male Wistar rats (200-300 g) were preserved with histidine-tryptophan-ketoglutarate (HTK) solution for 18 hours by simple cold storage (CS) or subjected to short-term resuscitation (STR) with oxygenated (pO(2) > 500 mm Hg) machine perfusion with HTK at 4 degrees C, 12 degrees C, or 22 degrees C for 2 hours with subsequent CS for 16 hours at 4 degrees C. Upon reperfusion in a normothermic perfusion circuit, STR significantly improved enzyme leakage (alanine aminotransferase, glutamate dehydrogenase) and metabolic recovery (tissue levels of ATP) providing best values at 12 degrees C or 22 degrees C. Moreover, a hugely increased gene expression of the adhesion molecule ICAM-1 as well as major histocompatibility complex (MHC) class II antigen was seen after CS, but significantly alleviated by STR at 4 degrees C or 12 degrees C. However, mRNA for both surface proteins rose significantly after STR at 22 degrees C compared with CS. In conclusion, STR by oxygenated perfusion is beneficial to the predamaged graft, facilitating later transportation and supervision of the graft compared with continuous machine preservation. However, increased perfusion temperature should be recommended only up to the limit of 12 degrees C to prevent overactivation of surface antigen expression.

Exogenous superoxide dismutase prevents peroxynitrite-induced apoptosis in non-heart-beating donor livers.

OBJECTIVE: To investigate the role of oxygen free radicals in the induction of apoptosis in non-heart-beating donor (NHBD) livers, and if superoxide dismutase (SOD) ameliorates these alterations. METHODS: Rat livers were perfused via the portal vein with histidine/tryptophan/alpha-ketoglutarate solution from heart-beating donors (HBD) or 60-min warm ischemia from NHBD, with or without the addition of SOD. After 24 h, cold storage livers were evaluated by isolated reperfusion. RESULTS: NHBD showed significantly higher enzyme leakage and elevated portal venous pressure (PVP) versus HBD. Bile and total adenine nucleotides (TAN) were significantly decreased. Apoptosis was prominent in sinusoidal lining cells, coupled with strong nitrotyrosine staining (NTR). The concentrations of nitric oxide and lipoperoxides were largely increased. SOD medication reduced hepatic enzyme release by 30% and lipoperoxides by nearly 50%. Apoptosis and NTR were significantly decreased, and PVP was strikingly reduced to normal values. A 3-fold enhancement in bile production and 1.5-fold increase in TAN of the liver tissue were also observed. CONCLUSION: NHBD livers are prone to severe reoxygenation injury promoted by oxygen free radicals, massive nitrite oxide production and peroxynitrite-induced apoptosis within the sinusoids. Antioxidant medication with SOD should be considered as a useful means of preserving NHBD livers.

Reduced liver apoptosis after venous systemic oxygen persufflation in non-heart-beating donors.

Graft injury caused by warm ischemia in livers from non-heart-beating donors (NHBDs) strongly affects posttransplantation outcome and is associated with liver apoptosis, which is mediated by death receptors, such as Fas, a surface receptor of the tumor necrosis factor (TNF)-alpha family. The aim of this study was to test the ability of venous systemic oxygen persufflation (VSOP) to reduce apoptotic changes and Fas activation in the liver after warm ischemic insult in vivo. Livers of male Wistar rats were harvested 30 min after cardiac arrest from non-heart-beating donors (NHBD) with (NHBD + O2) or without (NHBD) application of gaseous oxygen during the cold storage period via the suprahepatic caval vein. After 24 h of storage in University of Wisconsin solution at 4 degrees C, viability of the livers was assessed upon isolated reperfusion in vitro. Conventional signs of tissue damage like enzyme release and bile production showed a significantly elevated nonspecific cell injury in the NHBD group. TUNEL staining revealed increased DNA fragmentation of sinusoidal endothelial cells in the NHBD group and more apoptotic hepatocytes than in the control group. All these alterations could be almost abrogated by the use of VSOP in the NHBD + O2 group. The immunohistochemical staining of Fas antigen expression showed a significantly elevated Fas receptor expression in the NHBD and NHBD + O2 groups, in accord with an eightfold increase of Fas receptor mRNA detected by real-time reverse-transcription polymerase chain reaction (RT-PCR). These results demonstrate that the postischemic apoptotic rate of sinusoidal endothelial cells in NHBD livers can be reduced by the use of VSOP. A significant improvement in liver integrity and viability was obtained with this technique, without influencing the expression of Fas expression.

The isolated perfused rat liver: standardization of a time-honoured model.

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms.A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function). The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs-Henseleit buffer, without albumin. The usual volume is 150-300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37 degrees C. Standardized markers should be used to allow comparison with other experiments.

Phase I evaluation of all-trans-retinoic acid in adults with solid tumors.

PURPOSE: Prompted by recent demonstrations that all-trans-retinoic acid (all-trans-RA) had efficacy in acute promyelocytic leukemia, a phase I trial of all-trans-RA was conducted to establish the maximum-tolerated dose (MTD) before phase II testing. PATIENTS AND METHODS: Forty patients with a histologic or cytologic diagnosis of malignancy other than leukemia were treated with single daily oral doses of all-trans-RA ranging from 45 mg/m2 to 200 mg/m2. Doses of all-trans-RA were escalated in the next cohort of patients until the MTD was determined if the preceding dose level was not associated with significant toxicity. RESULTS: Lung cancer was the most common type of tumor included in the study (26 cases) followed by head and neck squamous cell carcinomas (three cases), and squamous cell carcinoma of the skin (two cases); other miscellaneous solid tumors were also represented. Toxicities included cheilitis, skin reactions, headache, and nausea and vomiting, as well as transient elevations of liver enzymes and triglyceride levels. Skin toxicities, consisting of erythema with desquamation and paronychia, were considered to be the dose-limiting toxicity, and were observed in two of six patients who received 175 mg/m2/d, and in two of five patients who received 200 mg/m2/d. Of the 30 patients with assessable lesions, response was evaluated in 26 patients and no major objective tumor response was observed. Two patients were able to receive the drug for longer than 1 year without significant toxicities. There was considerable variation in individual patients' peak plasma all-trans-RA levels, and a decrease in the area under the curve of all-trans-RA plasma concentration was observed in all four patients evaluated. CONCLUSION: For phase II study of adult patients, we recommend 150 mg/m2 of all-trans-RA administered orally once a day. However, for better optimization of drug administration schedules, further studies are needed.

Oxygenated machine perfusion preservation of predamaged kidneys with HTK and Belzer machine perfusion solution: an experimental study in pigs.

The objective of the present study was to evaluate the recently proposed aerobic machine preservation with the noncolloidal HTK solution by comparison with the colloidal Belzer machine perfusion solution (MPS) after procurement of marginal kidneys from non-heart-beating donors. Kidneys were harvested 40 minutes after cardiac arrest in German Landrace pigs and subjected to 18 hours of oxygenated hypothermic machine perfusion with either Belzer MPS or modified HTK via the renal artery (Psys < 40 mm Hg). During machine perfusion transrenal flow was approximatively twofold higher and calculated oxygen uptake was increased by 30% using the colloidal Belzer MPS, but overall enzyme release was comparable in both groups. After heterotopic transpantation with bilateral nephrectomy of the recipient, there were no differences with respect to initial tissue perfusion in vivo (evaluated by laser Doppler flowmetry) as well as urine production and median serum levels of urea or creatinine over 1 week of follow-up between grafts perfused with HTK or Belzer MPS. In conclusion, provision of oxygen during storage is possible by low-flow perfusion with HTK as with Belzer MPS.

Poststress glucose mitigates behavioral impairment in rats in the "learned helplessness" model of psychopathology.

Three experiments examined the effects of poststress glucose treatment in the learned helplessness model of psychopathology in rats. In experiment 1, rats were given access to water or 40% aqueous glucose immediately following exposure to inescapable tailshocks or simple restraint in a 2 x 2 factorial design. Inescapably shocked rats failed to drink the glucose solution during the poststress interval and failed to show any improvement 24 hours after stress induction in shuttle-escape performance. Consequently, all rats received preexposure to a sweetened glucose cocktail in an attempt to increase poststress ingestion following inescapable shock treatment in experiment 2. Under these conditions, poststress intake of the glucose cocktail eliminated behavioral impairment in inescapably shocked rats relative to water-treated shocked rats and water- and glucose-treated restrained controls. Experiment 3 demonstrated that glucose prophylaxis occurs in the absence of sucrose when rats are preexposed to a 40% glucose solution prior to stress induction.

Learned helplessness: an experimental model of the DST in rats.

Elevated ratings of anxiety and agitation in Dexamethasone Suppression Test (DST) nonsuppressors suggest a role for psychological stress in the generation of the hypothalamic-pituitary-adrenal cortical (HPAC) abnormalities characteristic of depression. We employed the learned helplessness model of depression to test the effectiveness of psychological stress in inducing a resistance of plasma corticosterone levels to dexamethasone suppression. Inescapably shocked rats exhibited corticosterone levels that were significantly more resistant to dexamethasone suppression than were the levels of rats receiving an equivalent amount of escapable shock or no shock. These results confirm the hypothesis that HPAC resistance to dexamethasone suppression is enhanced by the distress associated with the inefficacy of behavioral coping responses. The present findings represent the first analog of the DST in the learned helplessness model of depression. This DST model allows investigations into neurobiological mechanisms underlying the HPAC alterations in depression.

Reduction of oxidative tissue injury and endothelial dysfunction by graduated reperfusion after cardioplegic arrest in isolated rat hearts.

The aim of this study was to investigate whether or not a graduated resumption of the perfusion pressure after cardioplegic ischaemic arrest will reduce the impact of oxygen free radicals on myocardium and the cardiovasculature. Langendorff-perfused rat hearts were subjected to cardioplegia and subsequent 40 min of global ischaemia at 25 degrees C. Reperfusion was carried out either abruptly (AR) or gradually (i.e., perfusion pressure stepwise increased from 40 to 75 mmHg within 30 min -GR). GR resulted in a significant improvement of percentage recovery of left ventricular systolic pressure as compared to AR. A marked increase of thiobarbituric acid reactive substances (TBARS) was detected in the effluent during AR, accompanied by an impaired release of the endothelial vasodilator NO and diminished coronary flow rates compared to the baseline values. GR resulted in a significant reduction of TBARS in the effluent and promoted a better recovery of coronary flow as well as endothelial release of NO during the later phase of reperfusion. It is concluded that graduated reperfusion is beneficial in reducing free radical mediated peroxidative tissue injury and endothelial dysfunction upon reoxygenation.

Absence of association of HTLV-I infection with type 1 neurofibromatosis in the United States or Japan.

An HTLV-I tax transgenic mouse model develops a syndrome with similarities to type 1 neurofibromatosis (NF-1). To investigate possible associations between this human retrovirus and NF-1, we have analyzed 67 neurofibromas from Japan (where HTLV-I infection is endemic) and compared them with 21 cases from the United States. We were not able to identify virus in tumor tissue in either group. This suggests that HTLV-I infection is not commonly associated with NF-1.

Synthesis of high energy phosphates during cold ischemic rat liver preservation with gaseous oxygen insufflation.

The depletion of biochemical energy stores during anoxic ischemic preservation is a major problem affecting the viability of the graft in transplantation medicine. After cessation of blood flow and, thus, lack of metabolic substrates and oxygen supply, a swift decrease of energy-rich phosphates can be observed in the tissue, since endergonic metabolic processes continue, but no further oxidative regeneration of biochemical energy stores will take place. We investigated the effect of a continuous gaseous oxygen supply via the venous vessels during extended ischemic preservation of rat livers in University of Wisconsin preservation solution for 48 hr. Results showed that aerobic ischemic storage not only prevented the depletion of biochemical energy stores, but promoted a de novo synthesis of high energy phosphates, and significantly enhanced the functional recovery of the organs after postischemic reperfusion. The findings suggest that maintenance of oxidative energy metabolism largely protects the organ during ischemia and may enable organ viability even after extended preservation times.

Enhancement of endogenous cyclic AMP signal: a new approach to allow for cold preservation of rat livers from non-heart-beating donors?

BACKGROUND: The organ donor shortage has led to a reconsideration of the use of non-heart-beating donors (NHBDs). However, graft injury due to warm ischemia in NHBD livers strongly affects posttransplant outcome. The present study was aimed at investigating the role of the cellular cyclic (c)AMP second messenger signal with regard to hepatic viability after cold preservation of NHBD livers. METHODS: Cardiac arrest was induced in Wistar rats by frenotomy of the anesthetized nonheparinized animal. After 30 min, the livers were excised and flushed with 20 ml of heparinized saline solution, rinsed with 10 ml of University of Wisconsin (UW) solution, and stored submerged in UW solution at 4 degrees C for 24 hr. In half of the experiments, UW solution was supplemented with glucagon (0.5 microg/ml) to increase the cAMP signal in the liver. Reperfusion was carried out in vitro after all livers were incubated at 25 degrees C in saline solution to replicate the period of slow rewarming during surgical implantation in vivo. RESULTS: Hepatic levels of cAMP (nmol/g dry weight) declined from 1.21+/-0.05 to 0.53+/-0.03 (P<0.01) at 30 min after cardiac arrest. Subsequent storage in UW solution resulted in a further decline to 0.35+/-0.04 after 24 hr in group A, whereas glucagon treatment enhanced cellular cAMP signal to 0.64+/-0.06 (P<0.01). Upon reperfusion, liver integrity was significantly improved after glucagon administration, with 66% reduction in alanine aminotransferase release and a threefold increase in hepatic bile production as compared with untreated livers. Moreover, liver ATP tissue levels were restored to only 2.19+/-0.51 micromol/g in the untreated group but reached 4.97+/-0.41 micromol/g (P<0.05) after treatment with glucagon. CONCLUSIONS: Posthoc conditioning of predamaged livers by glucagon enhances cAMP tissue levels during ischemic preservation and improves hepatic integrity upon reperfusion. This may represent a promising approach for the use of livers from non-heart-beating donors in clinical transplantation.

Reduction of proteolysis by venous-systemic oxygen persufflation during rat liver preservation and improved functional outcome after transplantation.

An increase of cytosolic proteolytic activity during ischemic preservation and consecutive tissue degradation have recently been recognized as a major pathogenetic factor for liver injury during ischemia/reperfusion. In the present study, we propose a method for preventing proteolytic tissue disintegration, which results in improved recovery of the liver after transplantation. Livers were harvested from rats and stored for 24 hr at 4 degrees C in University of Wisconsin solution (group A). Others were additionally persufflated with gaseous oxygen via the inferior caval vein during this time (group B). At the end of ischemic preservation, proteolysis was confirmed in group A, with significantly elevated tissue levels of free alanine and free amino groups, whereas proteolysis was prevented in group B. After transplantation, the integrity of the graft was significantly improved in group B, in which there was a 50% reduction of plasma activities of alanine amino-transferase and a twofold increase in hepatic bile production after the onset of reperfusion, as compared with group A. Moreover, venous-systemic oxygen persufflation during cold preservation significantly attenuated the rise in plasma levels of malondialdehyde (MDA) after liver transplantation. In conclusion, venous-systemic oxygen persufflation during ischemic storage prevents tissue proteolysis and reduces parenchymal injury after transplantation in vivo; this technique may, thus, represent a useful adjunct in long-term liver preservation with University of Wisconsin solution.