Impact of polysol, a newly developed preservation solution, on cold storage of steatotic rat livers.

Chronic shortage of donor organs has led to acceptance of steatotic livers as grafts, although there is a higher risk of primary graft dysfunction. We herein report the beneficial impact of Polysol, a newly developed preservation solution, on cold storage of steatotic rat livers. Dietary hepatic steatosis was induced in Wistar rats by 2-day fasting and subsequent 3-day re-feeding with a fat-free, carbohydrate-rich diet. Fatty livers were retrieved, flushed and then stored at 4 degrees C for 24 hours with either HTK or Polysol. Functional integrity of the grafts was evaluated by isolated reperfusion with oxygenated Krebs-Henseleit buffer at 37 degrees C for 45 minutes in both groups. Polysol preservation resulted in significant reductions of not only parenchymal (AST (IU/L); 6728+/-824 in HTK vs. 3107+/-718 in Polysol; P < 0.001) but also mitochondrial (GLDH (IU/L); 3189+/-773 vs. 1282+/-365; P < 0.01) enzyme release throughout reperfusion. Moreover, PVP (16.9+/-2.7 vs. 7.8+/-1.5 mmHg; P < 0.05), hepatic O2 consumption (0.291+/-0.047 vs. 1.056+/-0.053 micromol/g liver/min; P < 0.001), tissue ATP content (0.695+/-0.086 vs. 1.340+/-0.157 micromol/g dry-liver; P < 0.005), bile production (0.79+/-0.11 vs. 4.08+/-0.66 microL/g liver/45-min; P < 0.001), malondialdehyde into the perfusate (1.922+/-0.198 vs. 0.573+/-0.094 nmol/L; P < 0.0001) and wet/dry-weight ratio of the liver tissues (5.20+/-0.31 vs. 3.85+/-0.15; P < 0.005) were all better preserved by Polysol. In line with these benefits, electron microscopy revealed that Polysol preservation substantially suppressed deleterious mitochondrial alterations in steatotic livers. In conclusion, cold storage using Polysol resulted in significantly better integrity and function of steatotic livers. Polysol, therefore, may be a new alternative especially for "marginal" organs.     

Experimental liver preservation with Celsior: a novel alternative to University of Wisconsin and histidine-tryptophan-alpha-ketoglutarate solutions?

Celsior, a low viscosity and low potassium preservation solution, has recently been tested successfully in the cold preservation of heart, lung, kidney and small intestine. The purpose of the present study was to evaluate the potential of Celsior in the cold preservation of the liver. Livers were harvested from male Wistar rats and then flushed with either Celsior (CE), University of Wisconsin solution (UW) or histidine-tryptophan-alpha-ketoglutarate solution (HTK) and stored for 24 h at 4 degrees C in the respective solution. The reperfusion was performed in vitro using a recirculating model with oxygenated (95% O(2), 5% CO(2)) Krebs-Henseleit buffer at 37 degrees C. To simulate the slow rewarming during the surgical implantation in vivo, all livers were stored for 30 min at room temperature prior to reperfusion. After ischemic storage and also after reperfusion some samples were freeze-clamped for analysis of tissue metabolites while others were tested for structural and functional integrity by the isolated perfusion. CE vs. UW vs. HTK: Metabolic preservation of tissue ATP (micromol/g dry weight) during cold storage was best with Celsior (0. 46 +/- 0.17 vs. 0.26 +/- 0.03 vs. 0.35 +/- 0.07; p < 0.05 CE vs. UW), but upon reperfusion energetic recovery was comparable in the three groups (3.45 +/- 0.66 vs. 4.27 +/- 0.41 vs. 3.63 +/- 0.64 micromol/g/dry weight). There appeared to be structural integrity during reoxygenation irrespective of the used preservation solution with comparable values of parenchymal enzyme release (ALT: 575 +/- 82 vs. 547 +/- 106 vs. 593 +/- 38 mU/g/l), bile production (18.0 +/- 1.0 vs. 18.5 +/- 2.5 vs. 18.7 +/- 1.4 microl/g/ min), and the release of acid phosphatase, an indicator for activated Kupffer cells (89 +/- 13 vs. 90 +/- 5 vs. 123 +/- 21 mU/g/l) in this in vitro model. Vascular flow characteristics were approximated by the portal perfusion pressure, which tended to be elevated upon initial reperfusion in the UW group (8.4 +/- 0.6 mm Hg) compared to 6.6 +/- 1.0 and 7.3 +/- 0.4 mm Hg in Celsior and HTK, respectively. However, the pressure values decreased to the normal range even in the UW group with ongoing perfusion. The sensitivity of our model in detecting protective effects of the tested solution was confirmed by a negative control group of livers stored in Ringer's solution at 4 degrees C, yielding an impaired recovery which differed by one magnitude from the three other groups. Within the limits of an in vitro study it is concluded from these results that Celsior may become a suitable alternative for liver preservation and further studies including a transplantation in vivo are strongly encouraged.     

Protective effects of ambroxol in hypothermic liver preservation: a transplant study.

The bronchosecretolytic agent ambroxol added to histidine-tryptophan-ketoglutarate (HTK) solution has recently been shown to protect cold stored rat hepatocytes. The aim of the present study was to confirm these observations in a rat liver transplantation model. Before orthotopic liver transplantation, donor livers from 30 syngeneic Wistar rats were assigned to three groups (n = 10): (A) in situ flush (ISF) and 1/2-h cold storage (CS) with HTK solution, (B) ISF and 3-h CS with HTK, and (C) ISF and 3-h CS with HTK + 10(-3) mol/L ambroxol. The efficacy of the drug was evaluated by postoperative survival (> 14 days) and liver enzyme release (ALT), bile flow, histomorphological injury, and malondialdehyde (MDA) level in the grafts 15 min after reperfusion. After 1/2-h CS with HTK solution (A), 90% of the transplanted rats survived. In comparison with donor conditions, bile flow in the reperfused grafts decreased to 87 +/- 5.3%, whereas postoperative ALT levels slightly increased. After 3-h HTK preservation (B), the survival rate decreased to 60%, while ALT values markedly increased and bile flow after reperfusion declined to 82 +/- 6.6%. Ambroxol added to HTK solution (C) enhanced bile flow to 106 +/- 3.4%(p < .05), and reduced ALT and MDA levels and histomorphological injury of the transplanted livers, so that its beneficial effect in organ preservation has been confirmed in the transplant model. However, survival rate was not improved by the agent, probably because of the low cold ischemia tolerance of the Wistar rat livers used.     

Value of α glutathione S‐transferase for in vitro evaluation of preservation injury in normal and steatotic livers

Abstract Liver steatosis is frequently encountered at organ harvest and, although functionally in‐apparent in the donor, may seriously affect the functional recovery of the graft after ischemic preservation. The present study was aimed to investigate the diagnostic value of alpha‐glutathione S‐transferase (GST) in non‐schemic and ischemic livers with or without compensated steatosis. A histologically documented mild to moderate steatosis was induced in livers of male Wistar rats by fasting for 2 days and subsequent feeding of a fat‐free diet enriched in carbohydrates. Fatty livers (FL) were retrieved and perfused in vitro for 45 min either immediately or after ischemic preservation at 4°C in HTK solution. Effluate was collected during isolated perfusion and later analysed for liver specific enzymes, including GST. Normal livers (NL) were excised from healthy rats and underwent the same protocol. Non‐ischemic livers showed similar enzyme release (FL versus NL) for ALT or GLDH but significant differences in GST. After ischemic preservation of NL, enzyme release increased mildly with respect to the non‐ischemic reference values for ALT, remained unchanged for GLDH and rose substantially for GST. In FL, there was a more than 10‐fold increase in all parameters, being most pronounced for GLDH as a marker of mitochondrial damage. It is concluded that GST may discriminate between healthy and suboptimal steatotic livers prior to ischemia and that the release of GST upon postischemic reperfusion of normal livers proves to be the most sensitive indicator for hepatocellular injury. However, GST turned out to be less useful for the detection of postischemic reperfusion injury in steatotic grafts.     

Beneficial effect of pentoxifylline on microvesicular steatotic livers submitted to a prolonged cold ischemia

BACKGROUND: The deleterious effect of steatosis on transplanted livers is mainly related to a microcirculation impairment. We investigated the effect of preservation duration on the recovery of isolated perfused rat steatotic livers and tested the effect of pentoxifylline (PTX), known to have a beneficial effect on hepatic microcirculation. MATERIALS AND METHODS: Fatty rat livers were obtained using a diet able to induce an 80% to 100% microvesicular steatosis within 7 days. We studied the effect of the duration of preservation (12 hr, 18 hr, and 24 hr) on fatty and normal isolated perfused rat liver. PTX was added to University of Wisconsin solution during cold storage (30 mM/kg of weight) and at reperfusion (3 mM) (n=5 livers in each group). Lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, bile production, and vascular resistance were evaluated. The liver injury at the end of perfusion was assessed by optical and electron microscopy. RESULTS: For a 24-hr preservation period, fatty livers demonstrated increased enzymatic release (aspartate aminotransferase: 42+/-16 vs. 17+/-5 IU/L/g of liver, P<0.005; alanine aminotransferase: 32+/-13 vs. 13+/-3 IU/L/g of liver, P<0.005; lactate dehydrogenase: 1,207+/-497 vs. 291+/-195 IU/L/g of liver, P<0.001). Vascular resistance (0.32 vs. 0.15 cm H(2)O/min/mL, P<0.0005) and bile output (67+/-24 vs. 141+/-61 mg/g of liver, P<0.05) were decreased. Peliosis appeared after an 18-hr preservation period for fatty livers compared with a 24-hr preservation period for controls. All these negative effects were suppressed by PTX. CONCLUSION: Diffuse microvesicular steatosis became deleterious only after long preservation times (24 hr). PTX prevented this effect.     

Preservation of steatotic livers in IGL-1 solution.

A new Institut Georges Lopez (IGL-1) solution was used to preserve steatotic livers. Steatotic (obese [Ob]) and nonsteatotic (lean [Ln]) livers from Zücker rats (n = 16, 8 Ln and 8 Ob) were preserved for 24 hours at 4 degrees C in University of Wisconsin (UW) or IGL-1 solution, respectively, and then perfused ex vivo for 2 hours at 37 degrees C. Additionally, Ob and Ln livers (n = 16, 8 Ln and 8 Ob) were preserved in IGL-1 plus Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME). Hepatic injury and function (aminotransferases, bile production, bromosulfophthalein clearance), and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion injury, such as oxidative stress, mitochondrial damage, and vascular resistance, were studied. Nitric oxide (NO) production and constitutive and inducible NO synthase were also measured. Steatotic and nonsteatotic livers preserved in IGL-1 solution showed lower transaminases, malondialdehyde, glutamate dehydrogenase levels, and higher bile production than UW-solution-preserved livers. IGL-1 solution protected against oxidative stress, mitochondrial damage and the alterations in vascular resistance associated with cold ischemia-reperfusion. Thus, at the end of reperfusion period, aspartate aminotransferase levels in steatotic livers were 281 +/- 6 U/L in UW vs. 202 +/- 10 U/L in IGL-1 solution. Glutamate dehydrogenase was 463 +/- 75 U/L in UW vs. 111 +/- 4 U/L in IGL-1 solution, and oxidative stress was 3.0 +/- 0.1 nmol/mg prot in UW vs. 2.0 +/- 0.1 nmol/mg prot in IGL-1 solution. These beneficial effects of IGL-1 solution were abolished by the addition of L-NAME, which implicates NO in the benefits of IGL-1. In conclusion, IGL-1 solution provided steatotic livers with better protection against the deleterious effects of cold ischemia-reperfusion injury than did UW solution.     

Improved right heart function after myocardial preservation with 2,3-butanedione 2-monoxime in a porcine model of allogenic heart transplantation.

BACKGROUND: Right heart dysfunction is a major cause for early morbidity and mortality after heart transplantation. Experiments were designed to evaluate the influence of the calcium-desensitizing drug 2,3-butanedione 2-monoxime (BDM) on right heart function in a porcine model of heart transplantation. METHODS: Donor hearts of domestic pigs were arrested with BDM in Krebs solution (n = 7) and with BDM in Bretschneider's histidine-tryptophan-ketoglutarate (HTK) solution (n = 6). There were 2 control groups: University of Wisconsin (UW, n = 6) and HTK (n = 6). An isovolumic model was used in which the right ventricular volume was precisely controlled in vivo with an intracavitary high-compliance balloon. After 4 hours of ischemia, hearts were transplanted into recipients. After 1 and 2 hours of reperfusion, the right ventricular balloon volume was increased in 10-mL increments until right ventricular failure occurred and the developed pressures were recorded. RESULTS: Maximal right ventricular developed pressures were significantly different after 2 hours of reperfusion (UW: 35 +/- 13 mm Hg; HTK: 47 +/- 8 mm Hg; Krebs+BDM: 49 +/- 9 mm Hg; HTK+BDM: 50 +/- 6 mm Hg; P =.04). Hearts subjected to BDM could be loaded with a significantly increased volume after 1 hour and after 2 hours (UW: 57 +/- 10 mL vs HTK: 43 +/- 8 mL vs Krebs+BDM: 70 +/- 10 mL vs HTK+BDM: 67 +/- 15 mL; P =.002). Postischemic right ventricular enddiastolic compliance was significantly increased in groups treated with BDM after 1 hour (P =.02) and after 2 hours (P =.039). CONCLUSIONS: The drug BDM significantly improves right ventricular function in a heart transplantation model. The increase in volume load and developed right ventricular pressure achieved by BDM application would translate into a decreased risk of right ventricular failure after clinical transplantation.     

Preservation of pig liver allografts after warm ischemia: normothermic perfusion versus cold storage

Warm ischemia is known to induce substantial damage to the liver parenchyma. With respect to clinical liver transplantation, the tolerance of the liver to warm ischemia and the preservation of these organs have not been studied in detail. In isolated reperfused pig livers we proceeded according to the following concept: Livers were subjected to 1 or 3 h of warm ischemia. Subsequently, these organs were preserved by either normothermic perfusion or cold storage (histidine-tryptophan-α-ketoglutarate, HTK) for 3 h each. After storage, liver function was assessed in a reperfusion circuit for another 3 h. Parameters under evaluation were bile flow, perfusion flow, oxygen consumption, enzyme release into the perfusate (creatine kinase, glutamic oxaloacetic transaminase (GOT), lactic dehydrogenase, and glutamic pyruvic transaminase), and histomorphology. Damage to the liver was lowest after warm ischemia of 1 h. The results after cold storage were superior to those after normothermic perfusion (GOT: 3.2±0.3 and 2.6±0.2 U/g liver; cumulative bile production: 14.7±2.1 and 9.4±1 ml, respectively;P<0.05). In contrast, we found substantial damage at the end of reperfusion in livers undergoing 3 h of warm ischemia under both preservation techniques with severe hepatocellular pyknoses and essentially altered nonparenchymal cells. The results suggest that pig livers undergoing 1 h of warm ischemia and cold storage for 3 h with HTK solution may lead to functioning after transplantation.     

Comparison of histidine-tryptophan ketoglutarate and University of Wisconsin solutions as primary preservation in renal allografts undergoing pulsatile perfusion

INTRODUCTION: University of Wisconsin (UW) solution is the standard preservation solution for organ transplantation. Histidine-tryptophan ketogluatarate (HTK) solution has been used increasingly for kidney, pancreas, and liver transplantation. This study compared HTK and UW used during kidney procurement with subsequent pulsatile perfusion. METHODS: Between January and October 2003, 91 deceased renal and simultaneous kidney pancreas transplants were performed (UW, n = 41, and HTK, n = 50). There were no differences with regard to donor and recipient demographics or cold ischemia. RESULTS: Delayed graft function occurred in 3 (7%) of UW and 4 (8%) of HTK-preserved kidneys (P = NS). There were no significant differences between patient or graft survival. There was an anticipated difference between total preservative volumes used (HTK: 4.1 +/- 1.0 vs UW: 3.0 +/- 0.5; P < .005). CONCLUSION: UW and HTK appear to have similar efficacy in kidney preservation with pulsatile perfusion. HTK preservation solution can be used safely in conjunction with pulsatile preservation for cold storage of renal allografts.     

Maintenance of physiological coronary endothelial function after 3.3 h of hypothermic oxygen persufflation preservation and orthotopic transplantation of non-heart-beating donor hearts.

OBJECTIVE: The use of non-heart-beating donors (NHBD) might increase the number of grafts available for transplantation. Experiments on heart transplantation from NHBDs demonstrated the necessity for oxygenation during preservation to allow sufficient myocardial recovery. It has been shown that, after 16 min normothermic ischemia followed by 3.3-h hypothermic preservation, excellent myocardial and cardiovascular recovery is attained, if coronary oxygen persufflation (COP) is included in the preservation protocol. Here tests are presented on the recovery of coronary endothelium derived relaxation (EDR) of NHBD hearts after preservation including COP. METHODS: After 16 min normothermic ischemia, pig hearts were stored for 3.3 h at 0-1 degrees C in modified HTK plus COP (mBHTK+COP, n=6) or in two control groups without COP: (1) with mBHTK (n=6); and (2) with HTK (n=4). Following orthotopic transplantation and 3 h of reperfusion with full blood, coronary EDR was tested in vitro using Substance P (SP) under indomethacin for prostaglandin blockage. Additional tests were performed adding L-NIL to block the NO-production by iNOS or L-NNA to block total NO production. RESULTS: The EDR in percent of precontraction was 78 +/- 7% after mBHTK+COP and 77 +/- 20% (mBHTK) or 72 +/- 7% (HTK) in the controls without significant differences between the groups. Physiologic values of normal coronaries were 75 +/- 9%. L-NIL for blockage of NO-production by iNOS resulted in unchanged relaxations. After blockage of total NO production by L-NNA, the SP-induced dilation was significantly reduced to 58 +/- 8% (mBHTK+COP) and to 48 +/- 8% (mBHTK) or 55 +/- 13% (HTK) in the controls. CONCLUSIONS: Even after 16 min of warm ischemia followed by 3.3 h of preservation with gaseous oxygen persufflation, orthotopic transplantation, and reperfusion the endothelium derived coronary dilatation was unchanged from physiologic values and similar to the controls without COP. Blockage of NO production by L-NNA resulted in equal values of EDR with or without COP, while blockage of NO production by iNOS did not influence the EDR reaction. Thus COP preservation, which has been shown to allow excellent recovery of preserved NHBD hearts, caused no damage to the coronary EDR mechanisms.     

Impact of organ preservation using HTK for graft flush and subsequent storage in UW in rat kidney transplantation

BACKGROUND: In kidney transplantation, preservation has a significant influence on organ function. Since previous reports have indicated a benefit of combining histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solution, we evaluated the effects of initial flush with low viscosity HTK, followed by storage in UW. MATERIAL AND METHODS: Kidneys from inbred Lewis rats were procured using HTK or UW for initially perfusion and re-flushed after 30 min with either solution. In a third group, after perfusion with HTK, organs were re-flushed with UW. Organs were stored for 16-24 h (4 degrees C). Study parameters were high-energy phosphates, histology, apoptosis, recipient survival and urine excretion of 15-F2t -isoprostanes (oxidative stress marker). RESULTS: Prior to transplantation, tissue ATP/ADP concentrations were: HTK/UW > UW-only > HTK-only. In transplanted kidneys, histological damage was highest after preservation in HTK-only. Twenty-four hours after transplantation (24 h cold ischemia time - CIT), cleaved-PARP was most abundant using UW-only. 16 h of CIT resulted in higher urine concentrations of isoprostanes in the order HTK-only (368 +/- 308) > UW-only (157 +/- 105) > HTK/UW (67 +/- 26), and was lower in HTK/UW after 24 h of CIT (146 +/- 38) vs. UW-only (507 +/- 33 pg/mg creatinine). Survival (24 h CIT) was significantly reduced, and percentage of initial non-functioning (INF) kidneys highest in HTK-only (2.6 +/- 0.3 days, 100%), compared to UW-only (13 +/- 4.4 days, 75%) and HTK/UW (18.5 +/- 4.6 days, 33%). CONCLUSIONS: In long-term preservation, UW is superior over HTK. However, our results indicate that perfusion with HTK prior to storage in UW may improve the results of UW alone which is reflected by better survival, lower rate of INF, higher cellular energy conservation and a decrease of free radicals.     

Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation

BACKGROUND: University of Wisconsin (UW) solution is currently the standard preservation solution used for abdominal organ transplantation. This study assesses the efficacy of histidine-tryptophan-ketoglutarate (HTK) compared with UW in pancreas transplantation. METHODS: Between October 2002 and August 2003, 20 pancreas transplants were performed. Patients were divided into two groups: UW (n=10) and HTK (n=10). Donor and recipient demographics were similar in both groups. The mean cold ischemia time for both groups was 11 +/-3 hr. RESULTS: There was an anticipated difference between total preservative volumes used (HTK: 4.5 +/- 1.2 L vs. UW: 3.4 +/-0.8 L; P =0.03). Patient and graft survivals to date were 100% in both groups. Serum fasting blood glucose, peak amylase, and serial amylase levels remained comparable at all intervals posttransplantation. CONCLUSIONS: Within this range of cold ischemia time, UW and HTK demonstrate similar efficacy in pancreas preservation.     

The mechanism of injury in a steatotic liver graft during cold preservation

BACKGROUND: Fatty livers are more prone to primary nonfunction after transplantation. It is known that cell injury is strongly associated with alterations in the content and composition of membrane lipids. We assumed that plasma membrane (PM) fluidity, which is the most important property of the membrane, differed between fatty and normal livers. METHODS: The livers from obese and lean Zucker rats were flushed with cold Ringer's lactate and University of Wisconsin (UW) solution via the portal vein and preserved in cold UW solution for 24 hr. Histological examinations of electron microscopy were performed to investigate of sinusoidal lining cells (SLCs). PMs were isolated using a discontinuous density gradient of Percoll, and the lipid compositions were determined by chromatography. RESULTS: SLCs of fatty livers were markedly injured compared with control livers even after short preservation time. Moreover, many blebs were observed in the obese rats even after short preservation time. As for PM lipid composition, the cholesterol/phospholipid (PL) ratio of total PM was 0.14+/-0.03 in the obese rats and 0.21+/-0.03 in the lean rats (P<0.05). The relative proportions of polyunsaturated fatty acids among PLs in PM were 35.7+/-1.2% vs. 45.9+/-1.5% (P<0.0001). These results indicated that the fluidity of the PM in the obese rats is decreased after exposure to low temperatures. CONCLUSIONS: Our results suggest that steatotic livers from obese donors are more susceptible to cold preservation injury than livers without steatosis because of the severe deterioration of SLCs, and it is associated with PM fluidity even after short-term cold preservation.     

HTK-N, a modified HTK solution, decreases preservation injury in a model of microsteatotic rat liver transplantation

Background

Ischemia/reperfusion injury is an obstacle especially in steatotic livers, including those with steatosis induced by acute toxic stress. Recently, a modified histidine-tryptophan-ketoglutarate (HTK) solution, HTK-N, has been developed. This solution contains N-acetylhistidine, amino acids, and iron chelators. This study was designed to test the effects of HTK-N on preservation injury to rat livers after acute toxic injury.

Methods

Microvesicular steatosis was induced by a single dose of ethanol (8?g/kg BW). Livers were harvested and stored at 4?°C for 8?h with HTK or HTK-N before transplantation. Tissue and blood samples were taken at 1, 8, and 24?h after reperfusion to compare serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), standard histology, and immunohistochemistry for myeloperoxidase (MPO), caspase-3, and inducible nitric oxide synthase. Survival was compared after 1?week. For statistics, Analysis of Variance and t test were used.

Results

HTK-N improved survival from 12.5?% in HTK to 87.5?% (p?<?0.05). Furthermore, liver enzymes were decreased to 2?C75?% of HTK values (p?<?0.05). Necrosis and leukocyte infiltration and MPO, caspase-3, and iNOS expression after transplantation were decreased (p?<?0.05).

Conclusions

This study demonstrates that HTK-N protects liver grafts with microvesicular steatosis caused by acute toxic injury from cold ischemic injury better than standard HTK most likely via inhibition of hypoxic injury and oxidative stress and amelioration of the inflammatory reaction occurring upon reperfusion.     

Mechanism of primary graft non‐function in a rat model for fatty liver transplantation

Abstract We established a fatty liver model in rat suitable for the model of human liver with steatosis by cholesterol enriched chow, and investigated the mechanism of primary graft non‐function in fatty liver transplantation (LTx) using this model. Grafts with steatosis caused primary graft dysfunction after LTx following even short cold preservation; however, no significant difference was recognized in mitochondrial function of the graft during preservation. Morphological findings were not different at 1 h after reperfusion between non‐steatotic and steatotic livers. Focal necrosis of hepatocytes was seen and the sinusoidal endothelial cells were injured 24 h after reperfusion. In addition, the fluidity of the plasma membrane decreased in fatty liver. Our results indicate that deterioration of sinusoidal endothelial cells after reperfusion causes graft dysfunction in LTx of steatotic liver.     

The impact of liver preservation in HTK and UW solution on microcirculation after liver transplantation

Severe microcirculatory disturbances due to endothelial cell damage and leukocyte adherence during reperfusion of transplanted livers are considered to contribute to early graft failure. Since the degree of reperfusion injury after liver transplantation depends on the length of preservation time and the solution used for preservation, the aim of our study was to assess three solutions with respect to microvascular perfusion and leukocyte adhesion. Therefore, rat livers were stored up to 24 h in Euro-Collins (EC), University of Wisconsin (UW), or histidin-tryphtophan-ketoglutarate (HTK) solutions prior to orthotopic transplantation. The livers were studied in situ 60 min postoperatively using intravital fluorescence video microscopy. Using simple syringe flushing (10 ml), sinusoidal perfusion decreased below 50% in EC preserved livers after 8 h preservation, in HTK preserved livers after 16 h preservation, and remained higher than 70% in livers preserved in UW up to 24 h. Permanent adhesion of leukocytes was increased more rapidly in organs after 1, 8, 16, and 24 h preservation in HTK (16%, 15%, 34%, and 49.7% ± 4.7%) compared to those preserved in UW (15%, 18%, 17%; and 32.7% ± 3.3%; P < 0.05). Using a 10-fold volumn of the organ weight of HTK solution during the harvesting procedure, with an 8 min equilibration period, sinusoidal perfusion (39.6 ± 4.7%) and leukocyte adhesion (42.7 ± 3.1%) were not improved after 24 h. In contrast, equilibration with a volumn of approximately 40-times the liver weight improved sinusoidal perfusion (70.8% ± 2.7%; P < 0.01) and leukocyte adhesion (24.9% ± 3.1%; P < 0.01) significantly. Thus, using HTK solution, simple flushing prior to long-term cold storage resulted in microcirculatory disturbances when compared to UW solution. Larger volumns of HTK solution with an additional equilibration period of 8 min, however, reduced leukocyte adhesion and improved sinusoidal perfusion to a similar degree as UW solution.     

Evaluation of a novel cold storage solution (HBs) in a rat kidney transplant model.

We developed an improved solution for hypothermic storage (0-4 degrees C) of kidneys. The cold storage solution (HBS) was composed of macromolecules, high-energy cellular substrates, and a mixture of antiproteolytic amino acids, antioxidants, and anti-inflammatory compounds. The objectives in developing this solution were to achieve superior metabolic support of the kidney during cold storage and to protect against ischemic injury. Inbred Brown Norway rats, weighing 225-250 g, were subjected to orthotopic ultrarapid technique for kidney isotransplantation to minimize warm ischemia and to test the preservation process. The kidney was transplanted after 12 h of preservation. The animals were divided into three groups based upon the preservation solution utilized: HBS solution, HTK solution (Custodiol), and UW solution (UWS)(ViaSpan). Among the recipients, each group had two subsets. The first subset of animals was used to assess survival at 7 days as well as the reperfusion damage index (RDI) based on the macroscopic physical characteristics of the kidney at the time of transplantation. The second subset in each group was utilized to measure serum creatinine and blood urea nitrogen at 4 and 7 days, and histology at death or sacrifice. Mean +/- standard deviation (M +/- SD) was used for all parameters studied. The HBS solution showed significantly better protection at 12 h when compared to HTK and UW solutions. The reperfusion damage index (RDI) showed excellent preservation in the HBS (14 +/- 1), good preservation in UWS (13 +/- 1.5), and moderate preservation in the HTK (11 +/- 2) group. Histology was in concordance with the RDI, showing better histological findings with HBS and UW solutions than with the HTK group. Serum creatinine was significantly better in the HBS group when compared to HTK and UWS. Survival was statistically different, with 80% survival at 7 days in the HBS group, 20% survival in the HTK group, and 50% survival in the UWS group (p < .05). The HBS solution offered a new alternative for kidney cold storage with significantly better results when compared to the current gold standards of HTK and UW solutions in Brown Norway rats. This solution warrants further testing in other mammals.     

Liver preservation with HTK: salutary effect of hypothermic aerobiosis by either gaseous oxygen or machine perfusion

The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.     

Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate glutarate (HTK) and phosphatebuffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P<0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K+/Na+ rations in HTK-preserved livers than in UW-preserved livers (all data P<0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successuful liver preservation.     

Amylase levels in preservation solutions as a marker of exocrine tissue injury and as a prognostic factor for pancreatic islet isolation

Occurrence of primary graft nonfunction of pancreatic islets demands research for new methods of organ preservation during cold ischemia conditions. Digestive enzymes released during preservation injure the islets for subsequent rewarming and islet isolation processes. The aim of our study was to assess the amylase level in preservation solution as a marker of exocrine tissue injury, allowing the prognosis of islet yield and viability. The experiments undertaken on rats used three commercially available preservation solutions: ViaSpan (UW); Custodiol (HTK); and Euro-Collins (EC). After 180 minutes of cold ischemia, the highest islet recovery was observed among pancreata stored in UW solution (508 +/- 139 vs HTK 344 +/- 103; P <.05 vs EC 322 +/- 113; P <.05). These islets also revealed the highest insulin stimulation index in glucose static tests (1.19 +/- 0.30 vs HTK, 0.87 +/- 0.43; P <.01, vs EC.25 +/-.06; P <.001). The highest amylase level in the preservation solution was associated with a decreased yield of islets during the isolation process and lowest insulin stimulation index (increasing 139 +/- 18% for EC, 108 +/- 12% for HTK; P <.05 vs 87 +/- 10% for UW; P <.05). Our data strongly suggest, that the dynamic of amylase release during pancreas preservation at 4 degrees C correlates with a reduced number and viability of isolated islets. These results suggest that measurement of amylase levels after pancreas preservation may have potential clinical application as a marker to evaluate pancreatic tissue injury.