Function of Cadaver Renal Homografts in Relation to Age of Donor,Cause of Death,and Ischaemia Time

Twenty-six renal grafts four months or more after transplantation have been shown to function satisfactorily when the donor''s age has varied from 10 to 64 years and the period of warm ischaemia has been between 26 and 133 minutes. It is suggested that a more lenient view be adopted with respect to the age of the donor, but that a warm ischaemia time of two hours should not be exceeded.     

Influence of Pre-, Intra- and Post-Operative Parameters of Donor Liver on the Outcome of Isolated Human Hepatocytes

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells. In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of extracellular glutathione in livers of anaesthetized rats by microdialysis with on-line high-performance liquid chromatography

An on-line analytical system for the continuous in vivo monitoring of extracellular glutathione (γ-glutamylcysteinylglycine)(GSH) concentrations in the livers of anaesthetized rats was developed. Microdialysates perfused through implanted microdialysis probes were collected with a loading loop of an on-line injector for direct and automatic injection into a high-performance liquid chromatographic system equipped with an electrochemical detector with AuHg electrodes. This method shortened the analysis time and circumvented the sample preparation process which is essential for accurate determination of GSH levels in biological samples. Additionally, this method provided continuous and real-time monitoring of extracellular GSH levels. Basal extracellular GSH concentrations in the livers of anaesthetized rate were found to vary over a wide range (from 4.16 to 76.5 μM). The method was applied to study the effect of global liver ischaemia on extracellular GSH concentrations and it was found that extracellular GSH levels in livers increased immediately with the onset of ischaemia and remained elevated for the 30-min ischaemic period. Ensuing reperfusion did reduce the GSH increase; however, the GSH levels did not return to the basal value.     

Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa

The use of banked human tissue, obtained with informed consent after elective surgical procedures, represents a powerful model for understanding underlying mechanisms of diseases or therapeutic interventions and for establishing prognostic markers. However, donated tissues typically have varying times of warm ischaemia in situ due to blood arrest or cold ischaemia due to procurement and transportation. Hence, before using these tissues, it is important to carry out pre-analytical studies to ensure that they are representative of the in vivo state. In particular, tissues of the gastrointestinal tract have been thought to have low RNA stability. Therefore, this study aimed to determine if extended warm or cold ischaemia times and snap-freezing or banking in RNA stabilization solution affects RNA integrity or gene expression in human ileum mucosa. In short, ileum mucosa was collected for up to 1.5 h and 6 h of simulated warm or cold ischaemia respectively. Subsequently, RNA integrity and gene expressions were determined. It was found that RNA integrity remained high over the course of warm and cold ischaemia examined and there were in general no significant differences between snap-freezing and banking in RNA stabilization solution. Following the same trend, there were in general no significant changes in gene expressions measured (MYC, HIF1α, CDX, HMOX1 and IL1β). In conclusion, RNA in the ileum mucosa is maintained at a high integrity and has stable gene expression over the examined time course of warm or cold ischaemia when banked in RNA stabilization solution or snap-frozen in liquid nitrogen. As the average warm and cold ischaemia times imposed by surgery and the process of tissue banking are shorter than the time period examined in this study, human ileum mucosa samples collected after surgeries could be used for gene expression studies.     

Effect of a transitory ischaemia on the structure-linked latency of rat liver acid phosphatase and beta-galactosidase.

The structure-linked latency of acid phosphatase and beta-galactosidase was studied in rat liver lobes made ischaemic for 1 or 2 h and then recirculated with blood for increasing periods. Free activity of acid phosphatase and unsedimentable activity of beta-galactosidase are increased in homogenates of ischaemic livers. When ischaemia had been maintained for 1 h, the recovery of normal latency for both enzymes was observed 1 h after re-establishment of the blood flow. After a 2 h period of ischaemia, unmasked activity markedly decreases during the first 1 h after restoration of blood flow; after that, a large and irreversible secondary rise takes place. Chlorpromazine, injected 30 min before or just after induction of ischaemia, extensively prevents the latency decrease occurring during restoration of blood flow. Modifications of the hydrolase distribution pattern obtained after differential centrifugation are in agreement with the latency changes. These results suggest that a 2 h ischaemia causes an alteration of the liver lysosomes that is largely reversible and that restoration of blood flow induces an irreversible alteration of these organelles. Chlorpromazine treatment prevents the irreversible lesion from taking place.     

Ischaemic preconditioning modulates the activity of Kupffer cells during in vivo reperfusion injury of rat liver

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

Ischaemia and tissue pyruvate dehydrogenase activities in the rat: a comparison of the effects of cervical dislocation and pentobarbital anaesthesia

The work defined the relationship between [long-chain acylcarnitine] and PDHa activities in hearts, kidneys and livers of rats sampled after cervical dislocation or pentobarbital anaesthesia. Although tissue [long-chain acylcarnitine] correlated with fatty acid availability or its mitochondrial oxidation in anaesthetized rats, this was not the case for hearts or kidneys of rats sampled after cervical dislocation. Cardiac [long-chain acylcarnitine] and PDHa activities were higher in rats killed by cervical dislocation. Metabolite changes within the hearts were consistent with tissue hypoxia and the effects of cervical dislocation were mimicked in hearts of pentobarbital-anaesthetized rats by 20s ischaemia. Renal and hepatic PDHa activities were unaffected by this short period of ischaemia. The susceptibility of cardiac PDHa to hypoxia or ischaemia may explain the variability in activities often observed within or between laboratories.     

Allopurinol Inhibits Lipid Peroxidation in Warm Ischaemic and Reperfused Rabbit Kidneys

Rabbit kidneys were subjected to 120min of warm ischaemia or to 120min of warm ischaemia followed by 60min reperfusion with blood in vivo before being removed, homogenised and incubated at 37°C for 90min. Lipid extracts were obtained and monitored for Schiff base (fluorescence emission 400-450 nm, excited at 360 nm), thiobarbituric acid (TBA)-reactive material (emission 553 nm, excited at 515 nm) and diene conjugates (absorbance at 237 nm). Samples removed before incubation were assayed for reduced glutathione (GSH) and oxidised glutathione (GSSG) to provide an index of glutathione redox activity (GSH : GSSG). Allopurinol injected systemically i.v. (a) 15mins before kidneys were clamped. (b) 15mins before they were reperfused or (c) as two injections (before clamping and before reperfusion) significantly inhibited these biochemical markers of lipid peroxidation. Administration before reperfusion had a markedly more pronounced effect than when allopurinol was given before warm ischaemia only. It is concluded that allopurinol is probably effective because of its ability to inhibit xanthine oxidase and consequently lipid peroxidation during reperfusion rather than by preventing loss of purine nucleotides from hypoxic cells during ischaemia.     

Normothermic machine perfusion attenuates hepatic ischaemia-reperfusion injury by inhibiting CIRP-mediated oxidative stress and mitochondrial fission

Extracellular cold-inducible RNA-binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia-reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL-6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission-related proteins Drp-1 and Fis-1. Further, NMP increased the mitochondrial biogenesis-related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP-preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL-7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP-mediated oxidative stress and mitochondrial fission.     

Desferrioxamine reduces susceptibility to lipid peroxidation in rabbit kidneys subjected to warm ischaemia and reperfusion

Rabbit kidneys were clamped and subjected to warm ischaemia for 60 or 120 min then reperfused with blood for 60 min or for 24 hr. Treated rabbits received desferrioxamine at 15 or 50 mg/kg i.v. 15 min before reperfusion. Their kidneys were then removed and assayed for phospholipid Schiff base fluorescence (ex. 360 nm, em. 435 nm), diene and triene conjugates by UV spectrophotometry (240 nm and 268 nm respectively), for superoxide dismutase and for reduced and oxidised glutathione to provide an index of glutathione redox activity. All indices of lipid peroxidation were significantly elevated in untreated rabbits and glutathione redox activity was reduced. Treatment with desferrioxamine however effectively prevented these deviations and in many cases maintained them at the levels in fresh rabbit kidneys. These data provide further evidence that lipid peroxidation occurring during the reperfusion period is superimposed on the damage set up during warm ischaemia and may be preventable by administration of suitable therapeutic agents.     

Assessment of a test of renal viability.

A test of renal viability using the uptake of 125I-iodohippurate by kidney biopsy specimens has been developed. It is applicable to all kidneys irrespective of the method of storage. The uptake of 125I-iodohippurate in experimental kidneys correlated well with warm or cold ischaemia time and subsequent renal function. The test was used for human cadaver kidneys offered for transplantation and there was good correlation between iodohippurate uptake and warm ischaemic time. With this test, prediction of renal function was accurate in 85% of human cadaver kidneys transplanted. Pulsatile perfusion had no effect on cadaver kidneys as measured by this test.     

Warm Ischemic Injury Is Reflected in the Release of Injury Markers during Cold Preservation of the Human Liver

Background

Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range.

Methods

Six human livers discarded for transplantation with variable warm and cold ischemia times were flushed at the end of preservation. The liver grafts were flushed with NaCl or Lactated Ringer’s, 2 L through the portal vein and 1 L through the hepatic artery. The vena caval effluent was sampled and analyzed for biochemical markers of injury; lactate dehydrogenase (LDH), alanine transaminase (ALT), and alkaline phosphatase (ALP). Liver tissue biopsies were analyzed for ATP content and histologically (H&E) examined.

Results

The duration of warm ischemia in the six livers correlated significantly to the concentration of LDH, ALT, and ALP in the effluent from the portal vein flush. No correlation was found with cold ischemia time. Tissue ATP content at the end of preservation correlated very strongly with the concentration of ALP in the arterial effluent (P<0.0007, R² = 0.96).

Conclusion

Biochemical injury markers released during the cold preservation period were reflective of the duration of warm ischemic injury sustained prior to release of the markers, as well as the hepatic energy status. As such, assessment of the flush effluent at the end of cold preservation may be a useful tool in evaluating suboptimal livers prior to transplantation, particularly in situations with undeterminable ischemic durations.     

Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation

Previous studies have indicated that pyruvate is able to reduce ischaemia/reperfusion (I/R) injury in a variety of tissues, but a full understanding of the effects is lacking. In this current preliminary study, magnetic resonance spectroscopy (MRS) was used to investigate the biochemical effects of differing concentrations of pyruvate (3 and 15mM) on liver metabolism during the cold hypoxic preservation period itself, in order to gain insight into possible mechanisms. Hepatic lactate, alanine, and succinate levels were increased in livers preserved with 15mM pyruvate added to the University of Wisconsin (UW) solution and were generally elevated (but to a lesser degree) in livers flushed with 3mM pyruvate, compared to those cold stored in UW alone. Further, from enzymatic assays of adenine nucleotides, 15mM levels of pyruvate were found to maintain higher ATP levels during short periods (up to 4h) of cold hypoxic storage than in UW stored livers, whilst energy charge ratios (after 4 and 24h) were also higher (P<0.01 in each case). This may arise from enhanced glycolysis secondary to an improved redox status in the pyruvate-treated livers, as evident by the increase in the levels of lactate.     

Impact of warm ischemic time on microsomal P450 isoforms in a porcine model of therapeutic liver resection

Human microsomes and hepatocytes obtained from non-transplantable livers of brain-dead donors are very useful in predicting the in vivo metabolism of xenobiotics in humans. Fresh liver specimens obtained from therapeutic liver resection are also useful for research in cases where non-transplantable livers are not readily available. In the present study, the effect of warm ischemic duration, in the course of hepatic surgery, on the activities of liver cytochrome P450 (CYP) CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A were evaluated in a porcine model. Partial occlusion (portal vein and hepatic artery occlusion) decreased the activities of CYP2C, CYP2E and CYP3A, but not those of CYP1A and CYP2D. CYP3A, known to account for an average 30% of total P450 content in the human liver was the most susceptible to the warm ischemia. These results demonstrate that the activities of CYP isoforms, particularly those of CYP3A, are markedly affected by warm ischemia; it is, therefore, essential that care should be exercised when using microsomes prepared from surgically removed livers.     

Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver.

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1‐10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.     

Measurement by HPLC of Desferrioxamine-Available Iron in Rabbit Kidneys to Assess the Effect of Ischaemia on the Distribution of Iron Within the Total Pool

A method for the determination of desferrioxamine-available iron in tissue fractions is described which involves incubation with desferrioxamine, extraction of desferrioxamine and its iron-bound form, ferrioxamine, and quantitation of these two forms of the drug by reversed-phase hplc analysis. Chelatable iron levels in the 1-10µMolar region could be accurately and reproducibly measured using this technique.

The desferrioxamine-available iron levels in both the cortex and medulla of rabbit kidneys were significantly elevated (up to 2-fold) after the organs had been subjected to 2 hours warm ischaemia or 24 hours cold storage at 0°C In hypertonic citrate solution. There was no change in the total iron content of the tissues under these circumstances and thus a redistribution of intracellular iron to more available pools had presumably taken place as a result of ischaemia. This redistribution of iron may be an important factor in the initiation of peroxidative damage to cell membranes upon reperfusion of the organ with oxygen.