Improved rat liver preservation by hypothermic continuous machine perfusion using polysol, a new, enriched preservation solution.

For experimental machine perfusion (MP) of the liver, the modified University of Wisconsin solution (UW-G) is most often used. In our search for an enriched MP preservation solution, Polysol was developed. Polysol is enriched with various amino acids, vitamins, and other nutrients for the liver metabolism. The aim of this study was to compare Polysol with UW-G for MP preservation of the liver. Rat livers were preserved during 24 hours with hypothermic MP using UW-G (n = 5) or Polysol (n = 5). Hepatocellular damage (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], alpha-glutathione-S-transferase [alpha-GST]) and bile production were measured during 60 minutes of reperfusion (37 degrees C) with Krebs-Henseleit buffer. Control livers were reperfused after 24 hours of cold storage in UW (n = 5). MP using UW-G or Polysol showed less liver damage when compared with controls. Livers machine perfused with Polysol showed less enzyme release when compared to UW-G. Bile production was higher after MP using either UW-G or Polysol compared with controls. In conclusion, machine perfusion using Polysol results in better quality liver preservation than cold storage with UW and machine perfusion using UW-G.     

Machine perfusion preservation of the pig liver using a new preservation solution, polysol

INTRODUCTION: The current gold standard for donor liver preservation is cold storage in a preservation solution (4 degrees C), such as Celsior or the University of Wisconsin solution (UW). Recent studies have suggested the benefits of machine perfusion (MP) over cold storage. To improve the results of MP, an enriched preservation solution (named Polysol) was developed, which in a rat liver preservation model proved to be superior to the UW-gluconate solution. The aim of this study was to assess Polysol in a pig liver preservation model. MATERIALS AND METHODS: Female pigs (35 to 40 kg) were used as liver donors. After heparinization, the liver was washed out using Ringer's lactate, followed by the preservation solution (4 degrees C). The liver was preserved for 24 hours by either cold storage using Celsior (n=5) or MP using Polysol (n=5). For analysis of liver damage and function, livers were reperfused for 60 minutes using oxygenated Krebs-Henseleit buffer. RESULTS: CS-Celsior caused significantly more damage compared with MP-Polysol (t=60, AST: 622+/-215 versus 222+/-55; ALT: 17+/-6 versus 5+/-1). Intravascular resistance during reperfusion was significantly higher after CS-Celsior compared with MP-Polysol (t=0, 0.20+/-0.01 and 0.11+/-0.02 mm Hg/mL/min, respectively). No differences were seen regarding ammonia clearance and urea production. In both groups, no bile was produced during reperfusion. CONCLUSIONS: In an ex vivo pig liver preservation model significantly less damage was observed after machine perfusion preservation using Polysol, in comparison to cold storage using Celsior.     

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.     

Verapamil improves rat hepatic preservation with UW solution

Verapamil, a calcium channel blocker, improves myocardial preservation during cold cardioplegia and protects against renal damage during periods of warm and cold ischemia. To determine if verapamil could prevent ischemic damage to livers during and after cold storage, harvested rat livers were flushed with either University of Wisconsin (UW) solution or UW solution with 25 mg/liter verapamil. Twenty rats were used in each group. After 24 hr of storage at 4 degrees C, livers were perfused with oxygenated blood through the portal veins for 2 hr at 37 degrees C and pH 7.4. Liver enzymes, electrolytes, and perfusate flow rate were determined at 30-min intervals. At 90 min of perfusion, the verapamil group of livers had less elevation of AST (110 +/- 17 IU/liter vs 172 +/- 25 IU/liter, P less than 0.05), ALT (115 +/- 21 IU/liter vs 210 +/- 34 IU/liter, P less than 0.05), and LDH (962 +/- 170 IU/liter vs 1452 +/- 253 IU/liter, NS). Verapamil livers produced more bile than controls (6.9 +/- 1.9 microliters/g vs 2.3 +/- 1.7 microliter/g, P less than 0.05) and maintained a higher portal flow rate throughout the perfusion. Both groups showed similar reduction in liver weights after storage (3.9 +/- 0.9% vs 2.8 +/- 0.7%) and required the same amount of bicarbonate for correction of acidosis during perfusion (2.6 +/- 0.2 mM vs 2.8 +/- 0.2 mM). Light microscopic exam after perfusion showed hepatocyte damage in 30% of control livers, but 0% of verapamil livers. We conclude that verapamil-treated rat livers showed less damage and better function upon reperfusion after 24 hr of cold storage. This agent may be clinically useful as an additive to the UW preservation solution for livers.     

Evaluation of rat liver apoptotic and necrotic cell death after cold storage using UW,HTK, and Celsior

BACKGROUND: The benefit of Celsior in liver graft preservation is controversial. In the isolated perfused rat liver model, we compared the effects of Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) preservation solutions on liver cell death. METHODS: Rat livers were stored at 4 degrees C for 0, 8, 16, or 24 hr in either Celsior, UW, or HTK and reperfused for 90 min (37 degrees C). Bile secretion and perfusate levels of liver enzymes and histone-associated DNA fragments were measured. Apoptosis and oncotic necrosis were analyzed in biopsies by DNA gel electrophoresis, hematoxylin and eosin histology, and enzyme histochemistry for lactate dehydrogenase (LDH) and 5'-nucleotidase (5'-NT). RESULTS: Perfusate flow rate through the liver during perfusion did not significantly differ among preservation solutions. Bile secretion was best preserved in UW livers after 16-hr (versus HTK livers) and 24-hr storage (versus HTK and Celsior livers). Enzyme leakage from UW livers was lower compared with HTK livers after 8-hr storage (serum glutamic oxaloacetic transaminase [SGOT], LDH) and with Celsior and HTK livers after 16-hr (SGOT, LDH) and 24-hr storage (SGOT, serum glutamic pyruvic transaminase, LDH, purine nucleoside phosphorylase). In situ LDH and 5'-NT activities were best preserved in UW livers (up to 24 hr), whereas enzyme activities declined remarkably in HTK livers (after 8 hr) and Celsior livers (after 16 hr of cold storage). Although perfusate DNA fragment levels were repeatedly lowest from Celsior livers, apoptotic DNA laddering and the number of fragmented nuclei in hematoxylin and eosin sections was not different among livers after 8, 16, or 24 hr of storage. CONCLUSIONS: Celsior and UW are equally effective in preventing rat liver cell death after 0-16 hr of cold preservation as compared with the less effective HTK solution. After 24-hr cold storage, rat livers were best preserved in UW. Furthermore, there was no significant difference in mode of cell death (apoptosis or oncotic necrosis) after storage in any of the three solutions.     

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.     

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.     

Functional recovery of donation after cardiac death liver graft by continuous machine perfusion preservation in pigs

Introduction

Grafts from donation after cardiac death (DCD) will greatly contribute to the expand the donor pool. However, these grafts may require the development of the preservation methods because of primary nonfunction and severe ischemic bile duct injury.

Methods

Porcine livers were perfused with a newly developed machine perfusion (MP) system. Each system for the portal vein or the hepatic artery had a roller pump, a flow meter, and a pressure sensor. The livers were perfused with University of Wisconsin (UW)-gluconate at 4°C-6°C for 3 hours after 2 hours simple cold storage (CS). The portal vein flow rate was 0.5 mL/min/g liver (pressure, 10 mm Hg) and the hepatic artery flow rate was 0.2 mL/min/g liver (pressure, 30 mm Hg). Orthotopic liver transplantation was performed in pigs comparing Group 1 (n = 4) procured after acute hemorrhagic shock preserved by MP, Group 2 (n = 3) procured after warm ischemia time (WIT) of 30 minutes with CS preservation, and Group 3 (n = 4) procured with 30 minutes of WIT and MP preservation.

Results

Collected effluent aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the perfusion solution and serum AST and LDH were significantly lower in Group 1. AST and LDH results were lower in Group 3 than Group 2. Survival rates in Groups 1 and 3 were 3/4, but 0/3 in Group 2.

Conclusion

MP preservation was a useful promising preservation mode for DCD liver grafts.     

Enzymatic and morphological evaluation of the hepatic cytoprotective effect of somatostatin and octreotide during extended experimental liver preservation

Cytoprotective effects on the liver of somatostatin (ST) and octreotide (OT) have been previously described in normothermic ischemia-reperfusion models. The purpose of this study was an enzymatic and morphological assessment of hepatic cytoprotective effects during extended cold storage. Rat livers were washed in situ via the portal vein with University of Wisconsin solution (UW) UW+ST, or UW+OT. After 24 or 48 hours of cold ischemia time (CIT), livers were reperfused for 2 hours via the portal vein with oxygenated KHB at 37 degrees C using a nonrecirculating ex situ isolated perfusion system. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine kinase (CK) were assessed in the perfusate during ex situ isolated reperfusion. After a 24-hour cold ischemia time (CIT) ALT, LDH, and CK levels were significantly lower (P < .05) in the UW+ST and the UW+OT livers than the UW livers. After 48-hour CIT, AST, ALT, LDH, and CK levels were significantly lower (P < .05) in the UW+ST and the UW+OT livers than the UW livers. Histopathological examination revealed mild differences after 24-hour CIT but an evidently less ischemically damage organ after 48-hour CIT. With the limitations of an in vitro model, ST and OT showed enzymatic and morphological effects during extended liver preservation.     

Fructose 1-6 bisphosphate versus University of Wisconsin solution for rat liver preservation: does FBP prevent early mitochondrial injury?

Background

Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers.

Methods

Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy.

Results

During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP.

Conclusion

FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury.     

[beta ]-Galactosidase as a marker of ischemic injury and a mechanism for viability assessment in porcine liver transplantation

Glycohydrolases are a group of enzymes contained predominantly within lysosomes, which are released during Kupffer cell activation or death. One of these, [beta ]-galactosidase, has been proposed as a marker of ischemia-reperfusion injury in the liver because Kupffer cell activation represents a primary event in the injurious reperfusion cascade. In this study, we compared B-galactosidase with more traditional indicators of liver injury and function in a porcine model of liver preservation. Porcine livers were allocated into two groups: group C (n = 5), preserved in University of Wisconsin solution by standard cold storage for 24 hours, and group W (n = 5), perfused with oxygenated autologous blood on an extracorporeal circuit for 24 hours. Both groups were subsequently tested on the circuit during a 24-hour reperfusion phase. The perfusate was sampled for levels of [beta ]-galactosidase, as well as traditional markers of liver injury and function. A sharp increase in [beta ]-galactosidase levels was seen on reperfusion of cold preserved livers to a level of 1,900 IU/mL. This contrasted dramatically with normothermically preserved livers, in which the level never exceeded 208 IU/mL (P = .002). [beta ]-Galactosidase levels showed much earlier and greater increases compared with transaminase levels in livers injured by ischemia. A rapid elevation in [beta ]-galactosidase levels corresponded well with poor liver function and more liver injury. Measurement of [beta ]-galactosidase is a simple test that quantifies ischemia-reperfusion injury of preserved livers. It is more sensitive than transaminases, with faster and larger increases in levels after ischemic injury. It can be useful in assessing the viability of a liver during machine preservation. (Liver Transpl 2002;8:21-26.)     

L-carnitine ameliorates abnormal vulnerability of steatotic rat livers to cold ischemic preservation

BACKGROUND: Up to 30% of all livers retrieved for organ transplantation exhibit steatotic transformations. Chronic organ-donor shortage has led to the acceptance of these organs for transplantation, although a higher risk of graft nonfunction is associated with the preservation of steatotic livers. METHODS: A dietary steatosis was induced in Wistar rats by fasting them for 2 days and feeding them with a fat-free diet. Fatty livers (n=14) were retrieved and flushed with 60 mL of histidine, tryptophane, alpha-ketoglutarate (HTK) solution. In half of the experiments, L-carnitine (5 mM) was added to the HTK. Functional integrity of the livers was evaluated by isolated reperfusion with KHB in a recirculating system at 37 degrees C for 45 minutes. RESULTS: Addition of L-carnitine to the HTK promoted a significant reduction of the enzyme leakage from the livers upon reperfusion. Release of alanine-aminotransferase was reduced to one third (127+/-22 vs. 423+/-61 U/L), and the loss of glutamate dehydrogenase in the perfusate could be reduced significantly (42+/-7 vs. 542+/-134 U/L) when compared with livers stored without additional medication. Morphologic corroboration of these data was obtained by electron microscopy. Although normal appearance of liver mitochondria was preserved at the end of the cold ischemic storage, reperfusion of cold-stored fatty livers entailed massive alterations and frequent destruction of hepatic mitochondria. However, these morphologic impairments were remarkably mitigated in the carnitine-treated group. CONCLUSIONS: L-carnitine represents a feasible metabolic adjunct for a safe and more successful preservation of ischemia-reperfusion-sensitive steatotic livers.     

Warm flush at 37 °C following cold storage attenuates reperfusion injury in preserved rat livers

Abstract Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4°C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8,16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 °C, 20 °C, or 37 °C and reperfused for 90 min (37 °C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme histochemistry (5′-nucleotidase and LDH). After 8-h CIT and a flush at 4°C, a release of endogenous HA (-7 %) was observed, whereas uptake of exogenous HA occurred after the 20°C flush (2 %, P = NS) and after the 37°C flush (24 %, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 °C (-10 %) that at 4 °C and 20 °C (-64 % and -17 %, respectively, p =0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 °C and 20 °C preflush groups, but not in the 37 °C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 °C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4°C or 37 °C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 °C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 °C before reperfusion, SEC and hepatocellular damage is attenuated.     

Novel short-term hypothermic oxygenated perfusion (HOPE) system prevents injury in rat liver graft from non-heart beating donor

OBJECTIVE: To assess a machine perfusion system in rescuing liver grafts from non-heart-beating donors (NHBD). SUMMARY BACKGROUND DATA: The introduction of extracorporeal liver perfusion systems in the clinical routine depends on feasibility. Conceivably, perfusion could be performed during recipient preparation. We investigated whether a novel rat liver machine perfusion applied after in situ ischemia and cold storage can rescue NHBD liver grafts. METHODS: We induced cardiac arrest in male Brown Norway rats by phrenotomy and ligation of the subcardial aorta. We studied 2 experimental groups: 45 minutes of warm in situ ischemia + 5 hours cold storage versus 45 minutes of warm in situ ischemia + 5 hours cold storage followed by 1 hour hypothermic oxygenated extracorporeal perfusion (HOPE). In both groups, livers were reperfused in a closed sanguineous isolated liver perfusion device for 3 hours at 37 degrees C. To test the benefit of HOPE on survival, we performed orthotopic liver transplantation in both experimental groups. RESULTS: After cold storage and reperfusion, NHBD livers showed necrosis of hepatocytes, increased release of AST, and decreased bile flow. HOPE improved NHBD livers significantly with a reduction of necrosis, less AST release, and increased bile flow. ATP was severely depleted in cold-stored NHBD livers but restored in livers treated by HOPE. After orthotopic liver transplantation, grafts treated by HOPE demonstrated a significant extension on animal survival. CONCLUSIONS: We demonstrate a beneficial effect of HOPE by preventing reperfusion injury in a clinically relevant NHBD model.     

TAT-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期炎性反应及肝功能的影响

目的 探讨HIV-1反式激活蛋白-血红素氧合酶-1(TAT-HO-1)融合蛋白对大鼠供肝冷保存期炎性反应及肝功能的影响.方法 成年健康雄性SD大鼠48只,体重250～300 g,开腹游离腹主动脉并结扎,于左右髂总动脉分叉处向心端穿刺腹主动脉,切断肝上下腔静脉,随机灌注4℃HTK液(C组,U=24)或4℃含TAT-HO-1融合蛋白50 μg/ml的HTK液(P组,n=24),至流出灌洗液清亮后停止灌注,取肝脏,置于相应保存液中冷保存.分别于冷保存即刻、6、12和18 h时取保存液并切取肝组织.采用全自动生化分析仪测定保存液谷草转氨酶(AST)、谷丙转氨酶(ALT)及乳酸脱氢酶(LDH)活性,免疫组化法检测肿瘤坏死因子α(TNF-α)的表达,光镜下观察肝组织形态学.结果 随冷保存时间延长,两组保存液AST、ALT及LDH活性升高,TNF-α表达上调(P<0.05);与C组比较,冷保存6、12、18 h时P组保存液AST、ALT及LDH活性降低,TNF-α表达下调(P<0.05).结论 TAT-HO-1融合蛋白可抑制大鼠供肝冷保存期的炎性反应,对肝功能具有一定保护作用.     

Markers of allograft viability in the rat. Relationship between transplantation viability and liver function in the isolated perfused liver

The relationship between transplant viability and liver function has been examined. Wistar rat livers were preserved at 4 degrees C for increasing intervals and then transplanted into Wistar rat recipients. Two critical times were identified, the longest preservation period with 100% transplantation success (4 hr) and the shortest preservation period with 100% transplant failure (8 hr). The comparable critical times were also identified in livers preserved at 37 degrees C (1 hr and 2 hr). Liver functions were studied by the isolated perfused liver technique in other rat livers stored at 4 degrees C or 37 degrees C for the critical times. Two liver function tests, AST and LDH concentration in perfusate, discriminated between viable and nonviable livers across as well as within preservation groups. AST gave the best separation between viable and nonviable livers. Some functions such as ALT concentration in perfusate separated viable from non viable allografts only within preservation groups. Other liver functions were more sensitive to preservation temperature than allograft viability. Oxygen consumption after cold preservation for either critical time was about twice control levels. Urea production was far below control levels in warm-preserved livers but almost normal in cold-preserved livers. Our results indicate that AST release into perfusate can be used as a screening technique to optimize preservation methods, reserving transplantation for confirming the most promising results.     

Survival transplantation of preserved non-heart-beating donor rat livers: preservation by hypothermic machine perfusion

BACKGROUND: Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. METHODS: This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. RESULTS: Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. CONCLUSIONS: These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.     

Rescue of the Cold Preserved Rat Liver by Hypothermic Oxygenated Machine Perfusion

The aim of the study was to investigate whether hypothermic oxygenated liver perfusion after cold liver preservation resuscitated metabolic parameters and whether this treatment had a benefit for liver viability upon reperfusion.
We preserved rat livers either by cold storage (UW) for 10 h, or by perfusion for 3 h (oxygenated modified UW) after 10 h cold storage. We assessed viability of livers after preservation and after ischemic rewarming + normothermic reperfusion ex vivo . Ten hour cold storage reduced mitochondrial cytochrome oxidase and metabolically depleted the livers. Oxygenated perfusion after cold storage resulted in uploaded cellular energy charge and oxidized mitochondrial cytochrome oxidase. Reperfusion after 10 h cold storage increased formation of superoxid anions, release of cytosolic LDH, lipid peroxidation, caspase activities and led to disruption of sinusoidal endothelial cells. In contrast, reperfusion after 10 h cold storage + 3 h hypothermic oxygenated perfusion resulted in no changes of lipid peroxidation, bile flow, energy charge, total glutathione, LDH release and of caspase activation, as compared to fresh resected livers.
This study demonstrates, that a metabolically depleted liver due to cold storage can be energy recharged by short-termed cold machine perfusion. The machine perfused graft exhibited improved viability and functional integrity.     

Glycogen storage of the liver: a determining factor of initial function of the hepatic graft]

In this study we have investigated the effects of hepatocytes glycogen storage on the quality of livers for transplantation. Rats were fed or fasted for 24 h and hepatocytes isolated and cold stored in UW solution for 24 and 48 hours. Viability of the cells was analyzed by LDH release after 2 hours incubation in L15 with O2. Also, rabbits were fed, fasted (48 h) or glucose fed (48 h) and livers cold stored for 6, 24 and 48 h in UW solution. Functions of the livers were analyzed by isolated perfusion for 2 hours. Hepatocytes from fasted rats released significantly more LDH than hepatocytes from fed rats after 24 and 48 h cold storage. In rabbit livers, fasting depleted glycogen by 85% but had no effect on ATP or glutathione concentration. Livers from fasted rabbits produced similar amount of bile, released similar concentrations of lactate dehydrogenase and aspartate transaminase into the perfusate, maintained similar concentrations of glutathione after 24 hours preservation when compared to fed animals. After 48 h preservation livers from fasted animals were less viable than livers from fed animals and the decrease of liver functions in livers from fasted animals preserved for 48 hours was prevented by feeding glucose. This study shows that liver glycogen storage in hepatocyte is an important metabolite for successful liver preservation. Glycogen may be a source for ATP and antioxydant synthesis during the early period of reperfusion.     

The effects of fasting on the quality of liver preservation by simple cold storage

Although livers can be successfully preserved for 24 hr or more, often the transplanted livers have poor or no (primary nonfunction) function. The quality of the liver does not appear dependent upon the time of preservation but may be dependent upon the condition of the donor. In this study we have investigated the effects of fasting on the quality of livers for transplantation. Rabbits were fasted (48 hr) and livers preserved in the UW solution for 6-8 hr. Functions of the liver were analyzed by isolated perfusion for 2 hr. Also, pigs were fasted for 72 hr, livers preserved for 12 hr, and viability determined by orthotopic transplantation. Fasting depleted the liver glycogen by 85% but had no effect on ATP or glutathione concentrations. Rabbit livers from fasted animals produced similar amounts of bile, released similar concentrations of lactate dehydrogenase (LDH) and aspartate amino transaminase (AST) into the perfusate, maintained similar concentrations of ATP and glutathione in the tissue, and had a similar intracellular K:Na ratio after 24-hr preservation when compared to livers from fed animals. After 48-hr preservation, livers from fasted animals were less viable than livers from fed animals, including: reduced bile production (2.0 +/- 0.3 vs. 5.0 +/- 0.9 ml/2 hr, 100 g), greater release of LDH (3701 +/- 562 units vs. 1123 +/- 98 units) and AST, less ATP (0.326 +/- 74 vs. 0.802 +/- 160 nmol/g), less glutathione (0.303 +/- 13 vs. 0.933 +/- 137 nmol/g), and a lower K:Na ratio (1.5 +/- 0.9 vs. 7.4 +/- 0.6). Pigs receiving livers from fed animals preserved for 12 hr had better survival (5/6, 83%) than livers from fasted animals (3/6, 50%). The results show that the nutritional status of the donor can affect the outcome of liver preservation and transplantation. Increased injury in livers from fasted animals may be due to the loss of glycogen that may be an essential source of energy in the initial posttransplant period. In clinical liver transplantation the nutritional status of the donor may be an important factor in the initial function of the liver, and methods to increase the nutritional status of the donor may be important in increasing the quality of livers.