In face, of the increasing shortage of donor organs for clinical transplantation, the donor acceptance criteria for liver retrieval have been expanded toward inclusion of older donors and the use of “less than optimal” grafts.
Although the quality of cold-stored livers slowly declines beyond approximately 12 hr of ischemia and the risk of primary dys- or nonfunction steadily increases (1–3), up to 20% of all liver transplantations were performed after cold ischemia times of more than 12 hr (2, 4).
However, in most cases, it is not evident at the time of liver retrieval whether the graft is going to endure extended times of cold ischemia. Successful reperfusion of ischemic tissue pivotally depends on an adequate redox and intracellular signal homeostasis.
Previous studies have raised the concept to improve the outcome of marginally preserved organ grafts by post hoc reconditioning even after extended times of conventional cold storage (5, 6). Both techniques for temporary hypothermic oxygenation, oxygenated machine perfusion, or gaseous oxygen persufflation were later shown to be equally effective to improve ulterior recovery on reperfusion in small animals (7), supposedly because of a beneficial influence on the mitochondrial redox homeostasis during conditions of cold- induced metabolic retardation (8). Owing to its simplicity and ease of application, gaseous oxygen persufflation seems to be particularly convenient for treatment of the liver.
In the rat, 90 min of endischemic oxygen persufflation had been demonstrated to improve organ integrity on subsequent reperfusion of normal and fatty livers (7, 8). Because the method is mainly operative by diffusion processes across interstitial fluid/parenchymal tissue and actual distances between oxygen-filled pathways, increase in proportion to organ size (9), restoration of redox homeostasis, and cellular ion balance by venous systemic oxygen persufflation (VSOP) might be a phenomenon, subject to different time courses in small or large organs. It is thus difficult to delineate the optimal persufflation time in larger organs.
The present investigations were aimed to clarify in a controlled large animal experimental setting to which extent short-term gaseous oxygen persufflation actually works to improve liver quality after long-term preservation and to which extent the physical properties of a larger organ affect the temporal kinetics to achieve an optimal effect.
Energetic Recovery During Hypothermic Reconditioning
Metabolic efficiency of hypothermic reconditioning (HR) was monitored by restoration of tissue levels of ATP at the end of ischemic preservation and varying times of subsequent VSOP (Fig. 1). It is seen that already 1 hr of HR led to a significant rise in liver ATP, which was further improved by 2 hr of HR. No notable additional effect could be evidenced by extending the reconditioning time to 3 hr.
It is to note that the extend of ATP replenishment provided by 2 hr of HR in the pig livers corresponded well to the effect seen in human livers, where 1 to 2 hr of oxygen persufflation after a mean preservation time of 12 hr resulted in an increase of liver ATP concentrations from 1.06±0.32 to 3.27±0.47 (partly taken from Ref. 10).
Liver Viability in Relation to the Time of HR
Liver enzymes were measured in serum samples taken at different time points during reperfusion and taken as a general indicator of hepatocellular injury (Fig. 2). Serum activities of aspartate aminotransferase were found elevated in the untreated group, and a progressive rise in enzyme leakage was observed during ongoing reperfusion. HR did result in a notable net reduction of hepatic enzyme loss, which was significant already after 1 hr of HR and maximal after 2 hr.
Similar results were also found with respect to the hepatic loss of lactate dehydrogenase, maximal values of which amounted to 1512±84, 908±109*, 825±70*, and 1017±101* U/L/g (untreated vs. HR1 vs. HR2 vs. HR3, respectively; *P< 0.05 vs. untreated).
Likewise, parenchymal function of the grafts was gradually influenced by HR. Cumulative production of bile juice, synthesis of cholinesterase, and mean hepatic oxygen consumption during 120 min of reperfusion were significantly increased by 2 to 3 hr of preceding oxygen persufflation, whereas this effect was less consistent after only 1 hr out of treatment (Fig. 3a–c).
With respect to these parameters, it was seen that maximal protection of the graft is achieved by 2 hr of reconditioning. Extension of the persufflation time beyond 2 hr did not add to the therapeutic effect. Therefore, we choose 2 hr of HR as the standard protocol for further investigations.
HR Improves Vascular Perfusion After Cold Storage
Elevated vascular resistance was observed in the portal and the arterial system on reperfusion of untreated cold-stored livers (Fig. 4). After a short period of postischemic reactive arterial hyper-perfusion in both groups, an abnormal fall in arterial vascular perfusion was seen in untreated livers but was prevented by HR. Postischemic vascular resistance on flow-constant perfusion of the portal vein was also increased in untreated livers but significantly normalized by HR.
HR Reduces Postischemic Inflammatory Response in Liver Grafts
Proinflammatory response to preservation injury was evaluated by the release of tumor necrosis factor (TNF)-α into the circulation and was thus found to be significantly alleviated by the reconditioning protocol (Table 1). Cellular surface activation and innate immune response Toll-like receptor (TLR)-4 could also be mitigated by oxygen persufflation, resulting in significantly reduced expression of TLR-4 in the liver tissue at 120-min postreperfusion and a trend for lower expression of von Willebrand factor (vWF) in reconditioned versus untreated grafts. The almost 50% reduction of vWF obtained by oxygen persufflation, however, did not reach statistical significance (P=0.13).
Additional evidence for a beneficial effect of HR on vascular surface activation is seen in the fact that trapping of thrombocytes in the reperfused graft was significantly reduced in the HR-group when compared with untreated livers (compare with Table 1).
The impact of oxygen persufflation on free radical- induced tissue alterations was approximated by fluorimetric detection of lipid peroxidation (LPO) in deproteinized liver homogenates. Actually, it is seen that tissue LPO at the end of reperfusion was similar in untreated livers and in grafts subjected to HR (16.4±3.1 nmol/g vs. 10.2±3.8 nmol/g, respectively; P>0.05).
Influence of HR on Hepatocellular Tissue Integrity
In line with the data on hepatocellular enzyme loss, hematoxylin-eosin histopathology after reperfusion of untreated grafts revealed moderate liver injury, which was characterized by partial disruption of the general architecture, vacuolization, and disrupted cell and organelle membranes (score 1.83±0.13). HR resulted in less severe alterations and exhibited better preserved hepatic morphology. Score values for necrotic tissue injury were thus reduced by 2 hr of HR to 0.99±0.30 (P<0.05 vs. untreated).
The main objective of this study was to establish evidence for the effectiveness of vascular oxygen persufflation for short-term reconditioning of large size organ grafts after ischemic preservation.
Because of the biophysical mechanisms of gaseous oxygen persufflation (9), it is arbitrary to extrapolate the optimal duration of persufflation from rat livers to larger size organs. Because temporal kinetics of diffusion driven tissue conditioning might be influenced by the size of the organ, extrapolation from rat liver experiments are subject to underestimate the necessary application time in larger grafts. Therefore, an additional focus was spent on the influence of different persufflation times on early graft function on reperfusion. It was found that already 1 hr of oxygen persufflation resulted in a notable amelioration of consecutive liver recovery, but the effect was most consistent after 2 hr of oxygen persufflation. Most importantly, further extension of the reconditioning time did not result in any additional improvement. Thus, the optimal time protocol for gaseous liver reconditioning does not differ significantly from previous results in rats (8) and lies within the limits of clinical applicability in the pretransplant setting. Moreover, the metabolic effect of 2 hr of HR on tissue energetics seemed to be comparable in porcine and human livers, suggesting that the results of this study are likely to be relevant to the clinical situation.
Cold ischemia is accompanied by an extensive decline of cellular energy resources as seen by the depletion of ATP tissue concentrations (11, 12), which sensitizes hepatocytes to rewarming and reperfusion injury (12). Resynthesis of high-energy nucleotides by oxygenated perfusion before transplantation has hence been seen as major cause for improved resilience against rewarming injury (5, 12). The enhancement of endischemic ATP concentrations, which are actually increased also by VSOP (7, 10), might however be rather a readout for improved mitochondrial redox and signal homeostasis than the pivotal beneficial aspect of hypothermic oxygenation before reperfusion (7).
Recent evidences suggest proteolytic degradation of autophagy regulatory proteins to be responsible for impaired mitochondrial function and subsequent hepatocyte death on anoxia/reoxygenation. Autophagy normally occurs at low rates in cells to perform homeostatic functions such as lysosomal degradation of defective proteins, injured organelles, or depolarized mitochondria. Ischemic injury to isolated hepatocytes has been shown to depress hepatocellular autophagy and that this lack of autophagic clearance is causative for postischemic cell death (13). Interestingly, postischemic loss of autophagy can be prevented in rat livers by 90 min of oxygen persufflation (8). From this, an improved functioning of the cellular quality control systems and enhanced mitochondrial function can be expected (13). Through restoration of mitochondrial integrity and consecutively improved energy replenishment on warm reperfusion oxygen persufflation is conjectured to counteract tissue necrosis.
Another major factor compromising postpreservation recovery of liver grafts has been shown to be impaired vascular conductivity, leading to impaired vascular reflow and eventually deteriorating graft integrity as a result of insufficient oxygenation (14–16).
Impaired vascular perfusion was corroborated in this study by increased portal perfusion resistance and reduced arterial flow rates, both of which were significantly improved by preceding oxygen persufflation. Better and earlier resumption of normal cellular function because of improved provision and substrates and oxygen to postischemic tissue will have contributed to the overall enhanced parenchymal recovery after oxygen persufflation. Notably, the recovery of tissue levels of energy-rich phosphates after revascularization has generally been acknowledged as a primary predictor of organ viability after ischemic preservation (17, 18).
Hepatic ischemia-reperfusion induces platelet-endothelial cell interactions in arterioles, sinusoids, and venules already during early reperfusion. Cold preservation of sinusoidal lining cells results in increased platelet adhesion and platelet activation (19). Adherent platelets seem to participate in the development of sinusoidal perfusion failure in the postischemic liver (20), and the degree of platelet adhesion is an important determinant in assessing outcome of liver transplants in human (21).
It has already been shown that VSOP significantly enhances ultrastructural integrity of hepatic endothelium on reperfusion (22), putatively by providing energetic support before resumption of shear stress during reflow. In this study, it was furthermore disclosed that trapping of platelets in the liver graft during the experiments was significantly reduced after HR compared with the untreated group.
Down-regulation of cellular surface activation by HR was also translated by the mitigation of TLR-4 expression. Activation of TLR-4, one of a subset of pattern recognition receptors that recognize danger-associated molecular patterns, is known to trigger a cellular injury cascade in response to ischemia-reperfusion (23). This may lead to the activation of cellular responses that are known to regulate the innate immune response (24). and to the production of TNF-α by sinusoidal endothelial cells (25). The importance of TLR-4 signaling in the donor organ for ischemia and reperfusion injury sequel after liver transplantation has recently been demonstrated in a murine knockout model (26).
The concept of HR is not confined to the technique of gaseous oxygen persufflation. Oxygenated machine perfusion seems to promote comparable protection in rat livers (7), by mechanisms, presumably relating to improvement of mitochondrial redox state rather than depending on energetic support or nutritive stimulation (27). More recently, 1 hr of hypothermic oxygenated perfusion has even been shown to improve hepatic primary function after transplantation of pig livers, retrieved after cardiac death of the donor animal (28). In comparison with machine perfusion, already proven effective in a clinical pilot study (29), gaseous oxygen persufflation recommends itself as simple and less cost-intensive alternative
In conclusion, the results of this study discloses a useful method to regenerate cellular energy-dependent pathways under conditions of relatively low metabolic workload, being operative to suppress injurious cellular activation cascades on early reperfusion likely to augment postischemic recovery of long-preserved liver grafts. Two hours of “a posteriori” treatment provide the maximal effect and are recommended for further investigations.
MATERIALS AND METHODS
All experiments were performed in accordance with the federal law regarding the protection of animals.
Twenty four female German Landrace pigs, weighing between 25 and 30 kg were premedicated with ketamine (90 mg/kg), xylazine (10 mg/kg), and atropine (10 μg/kg) administered intramuscularly 10 min before induction of anesthesia. General anesthesia was induced by midazolam (0.5 mg/kg), pancuronium (0.2 mg/kg), and fentanyl (12.5 μg/kg) administered intravenously and maintained after intubation by mechanical ventilation with isoflurane in air/oxygen.
Under general anesthesia, the liver was dissected free and then perfused by gravity (60 cm H2O) through the portal vein with 3 L of histidine-tryptophan-ketoglutarate (HTK) preservation solution at 4°C. Livers were then excised, the hepatic artery was flushed on the backtable with additional 100 mL of HTK, and the graft was stored overnight in HTK for 18 hr.
After static cold storage at 4°C, the grafts were randomly used for the experiments without further treatment (untreated) or subsequently subjected to varying times of HR. This consisted of 1, 2, or 3 hr of VSOP as detailed previously (10, 30). During oxygen persufflation, the liver is left continuously placed immerged in cold preservation solution in a beaker; a catheter that is needed for oxygen influx, is fixed with a purse string suture in the suprahepatic vena cava. The caudal caval vein was closed with a bulldog clamp. Medical grade gaseous oxygen was filtered and insufflated by the caval catheter at a pressure limited to 18 mm Hg. Using a small (27 gauge) syringe needle, 2 to 3 small pinpricks were set into the dilated postsinusoidal venules at the margin of each liver lobe that allow the gas to leave the microvasculature.
Isolated Liver Perfusion Model
Immediately before reperfusion, all organs were exposed to no flow conditions at room temperature for 30 min to simulate the period of slow rewarming of the graft during surgical implantation in vivo (31).
Graft integrity was tested thereafter by isolated reperfusion in vitro through portal vein and hepatic artery in a recirculation system for 120 min. The perfusion medium consisted of heparinized autologous blood retrieved from the donor during the organ recovery operation, preserved overnight in commercial whole blood bags. The blood was diluted with freshly prepared Williams E solution to a hematocrit of 20% before the experiment.
Livers were placed in a moist temperature chamber and perfused at 38°C. Circulating blood was oxygenated in a temperature-controlled hollow fiber oxygenator with Carmedia plasma resistant bioactive surface (Minimax Plus; Medtronic Inc., Minneapolis, MN). Gas flow to the oxygenator (air, oxygen, and CO2) was differentially regulated to achieve physiological blood gas values (pO2 ∼150–200 mm Hg; pCO2 ∼30–50 mm Hg). Temperature was regulated by a circulating thermostat, connected to perfusion chamber and oxygenator.
Hepatic artery perfusion pressure was set at 80 mm Hg and automatically maintained by servo-controlled roller pump, connected to a pressure sensor placed in the inflow line immediately before the arterial cannula.
Perfusion of the portal vein was performed in a flow-constant manner (1 mL/g/min) driven by a centrifugal blood pump (Bio-Pump BP-50; Medtronic Inc.), whereas portal venous pressure was recorded using a water column connected to the inflow tract (32). Erythrocyte count and hemoglobine values remained unchanged during liver perfusion over the whole 120 min of investigation. Baseline values immanent to the experimental model were obtained from four livers that were reperfused immediately after procurement without ischemic preservation.
Hepatic oxygen consumption (mL/100 g/min) was calculated from arterial, portal, and hepatic venous oxygen saturations, total hemoglobine, and oxygen partial pressures using the following equation, taking into account the respective flow rates and liver mass:
with SaO2, SpO2 and ScO2 oxygen saturations (%) in hepatic artery, portal vein, and caval vein, respectively; Hb is total hemoglobin (g/dL); ApO2, PpO2 and CpO2 oxygen pressure (mm Hg) in hepatic artery, portal vein, and caval vein, respectively; Qa and Qp are arterial and portal blood flow (mL/min), respectively; and LW is liver weight (g).
Serum enzyme activities of aspartate aminotransferase, lactate dehydrogenase, and cholinesterase were assessed photometrically, using commercialized standard kits (Fa. Roche, Mannheim, FRG).
Serum concentrations of TNF-α were analyzed at the end of the experiments using a pig-reactive ELISA test kit on a fluorescence microplate reader (Tecan, Grailsheim, Germany), according to the instructions of the manufacturer (R&D Systems, Wiesbaden, Germany).
Oxygen free radical-induced tissue injury was approximated by the degree of LPO in the tissue at the end of reperfusion. LPO was evaluated by fluorimetry from deproteinized liver samples using the adduct formation with thiobarbituric acid as detailed elsewhere (33).
Hepatic arterial and portal vascular perfusion resistance was calculated from independently measured flow and pressure values, normalized to liver mass and expressed in mm Hg/L/min/100 g.
The common bile duct of the livers was cannulated with polyethylene tubing. Bile was collected during the whole reperfusion period, and hepatic bile production was calculated as mL/kg/hr.
Tissue Extraction and Assay of High Energy Phosphates
Tissue specimen for assessment of energetic status after reperfusion was taken with precooled steel tongs, immersed in liquid nitrogen and stored at −80°C until later analysis. For analysis of endischemic tissue ATP after varying times of persufflation, additional experiments were performed without reperfusion. In these experiments, two samples per liver were taken from different parts of the liver, always avoiding proximity to the pin pricks.
High-energy phosphates were determined enzymatically in the neutralized supernatant after protein extraction with perchloric acid of freeze-dried tissue samples as described elsewhere (7). The results were corrected for the respective dry weight to wet weight ratio of the tissue samples and expressed as μmol/g dry weight.
Reverse-Transcriptase Polymerase Chain Reaction Analysis
Total RNA was isolated from snap-frozen samples using TRI reagent (Applied Biosystems, Darmstadt, Germany). Equal amounts of RNA were quantified by Nano Drop (Fisher Scientific, Schwerte, Germany) complementary DNA by incubation with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The polymerase chain reaction mix was prepared by using TaqMan GenEx Master Mix (Applied Biosystems). The amount of specific mRNA in the tissue was expressed in arbitrary units after normalization for the respective individual quantities of transcripts of glyceraldehyde phosphate dehydrogenase, which was analyzed as housekeeping gene. Primers for glyceraldehyde phosphate dehydrogenase (TaqMan Gene Expression Assay noSs03375435_u1) and TLR-4 (n°Ss03389780_m1) were purchased from Applied Biosystems.
Sequences of the polymerase chain reaction primers for vWF, customized by Applied Biosystems, were as follows: sense, ACTACGACCTGGAGTGTGTGA and antisense, CTGTTCTCATGCCGGACCAT.
Liver tissue was collected at the conclusion of the experiments, cut into small blocks (3-mm thickness), and fixed by immersion in 4% buffered formalin. The blocks were embedded in paraffin and cut into 2-μm sections using a microtome. Hematoxylin-eosin staining was used to judge morphological integrity of the parenchyma. Sections were examined at 200× magnification and the extent of necrosis graded semiquantitatively from 0 (no necrosis) to 3 (severe necrosis with disintegration of hepatic cords) as described elsewhere (34), in a blinded fashion by two independent investigators.
All values were expressed as means±standard error of the mean of n=6 animals per group. After proving the assumption of normality and equal variance across groups, differences among groups were tested by analysis of variance followed by the Student-Newman-Keuls test or Student's t test where appropriate. Statistical significance was set at P less than 0.05.
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