ORIGINAL ARTICLE

Correlation Between the Liver TemperatureEmployed During Machine Perfusion andReperfusion Damage: Role of Ca2�

Mariapia Vairetti, Andrea Ferrigno, Vittoria Rizzo,2 Eleonora Boncompagni,3 Amedeo Carraro,4

Enrico Gringeri,4 Gloria Milanesi,3 Sergio Barni,3 Isabel Freitas,3 and Umberto Cillo4

1Department of Internal Medicine and Therapeutics, 2Department of Biochemistry, Istituto di Ricovero eCurae Carattere Scientifico Policlinico S. Matteo, 3Department of Animal Biology and Consiglio Nazionaledelle Richerche-Instituto di Genetica Molecolare, University of Pavia, Pavia, Italy; and 4Department ofGeneral Surgery and Organ Transplantation, University of Padua, Padua, Italy

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, therole of Ca2� levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs-Henseleit (KH)solution (with 1.25 or 2.5 mM CaCl2) at 4°C, 10°C, 20°C, 25°C, 30°C, or 37°C. For cold storage, livers were perfused in situand preserved with Celsior solution at 4°C for 6 hours. The reperfusion period (2 hours at 37°C) was performed under the sameconditions used for MP-preserved and cold storage–preserved livers. Hepatic enzyme release, bile production, adenosinetriphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37°C caused marked enzymerelease; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significantdecrease in liver damage. High levels of biliary gamma-glutamyltransferase and lactate dehydrogenase were found with MPat 4°C and 10°C but not with MP at 20°C. When a KH–1.25 mM CaCl2 solution was used during MP at 20°C, very low enzymerelease was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparisonwith cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH–2.5 mMCaCl2. ATP levels were higher and morphology was better in liver preserved with KH–1.25 mM CaCl2. MP at 20°C withKH–1.25 mM CaCl2 resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, incomparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liverMP. Liver Transpl 14:494-503, 2008. © 2008 AASLD.

Received July 20, 2007; accepted October 10, 2007.

Maintaining liver viability during preservation is impor-tant for improving the outcome of organ transplanta-tion. During the past decade, various authors have re-ported the advantages of hypothermic machineperfusion (MP), including the use of organs from non–heart-beating donors, for increasing liver viability dur-ing organ preservation in comparison with conventionalcold storage.1-3 MP is usually performed at a hypother-

mic temperature even though current experience sug-gests that lowering the temperature results in vasocon-striction of the hepatic vasculature. It causes anincrease in flow resistance with disruption of hepaticmicrocirculation due to an imbalance in the productionof vasoconstrictor and vasodilator substances withinthe liver.4 Furthermore, in clinical liver transplantationand experimental models, reperfusion of liver grafts

Abbreviations: AST, aspartate aminotransferase; ATP, adenosine triphosphate; C, collagen fibers; D, bile duct; f, fibroblast; G,glycogen; �GT, gamma-glutamyl transpeptidase; I/R, ischemia-reperfusion; KH, Krebs-Heinseleit; L, lipid droplets; LDH, lactatedehydrogenase; m��, transmembrane potential; m, (myo)fibroblast; M, mast cell; MP, machine perfusion; NAC, n-acetyl-cysteine; P,portal vein; RR, ruthenium red; UW, University of Wisconsin; UW-G, University of Wisconsin–gluconate.Supported by Ministero dell’Universita e della Ricerca 2004 and 2006 (through the project “Normothermic and SubnormothermicMechanical Perfusion vs Traditional Hypothermia at 4°C in Hepatic Preservation for Transplantation: Complex Analysis by IntegratedBioanalytical Methods on Animal Models and on Human Livers”) and by Fondo d’Ateneo per la Ricerca (University of Pavia).Address reprint requests to Mariapia Vairetti, Ph.D., Department of Internal Medicine and Therapeutics, University of Pavia, Piazza Botta 10, 27100Pavia, Italy. Telephone: �39 0382 986346; FAX: �39 0382 28426; E-mail: vairetti@botta.unipv.it

DOI 10.1002/lt.21421Published online in Wiley InterScience (www.interscience.wiley.com).

LIVER TRANSPLANTATION 14:494-503, 2008

© 2008 American Association for the Study of Liver Diseases.

after cold preservation is associated with diminishedbile production.5,6 In fact, cholangiocytes play a sub-stantial role in damage caused by preservation underhypothermic conditions: compared with parenchymalcells, these cells are particularly susceptible to injuryinduced by cold hypoxia.7 No data exist to correlate thetemperature used during MP preservation and reperfu-sion damage. The shortage of organs dictates the use ofmarginal organs such as those containing fat due toalcohol or obesity: these organs are more susceptible tocold preservation because the plasma membrane andthe relative proportions of polyunsaturated fatty acidsdiffer between fatty and normal livers, and this showsthat the fluidity of the plasma membrane in obese ratsis decreased even after short-term cold preservation.8

Furthermore, at temperatures below 18°C, lipids un-dergo a transition into a gel phase or into other 3-di-mensional crystalline structures.9 Moreover, hypother-mic preservation of organs reduces the production ofadenosine triphosphate (ATP) by mitochondria, whichlimits the extrusion and/or storage of cell Ca2� by ATP-dependent pumps. Hypothermia also induces an imbal-ance in the cell’s electrical potential that can lead to anuncontrolled influx of Ca2� through plasma membraneCa2� channels.10 Moreover, reactive oxygen intermedi-ates released during reperfusion10,11 are known tocause Ca2� influx.12 Recently, some reports have dem-onstrated that mitochondrial Ca2� accumulation oc-curs at low ATP concentrations because of not onlyuniporter deinhibition but also the stimulation of mito-chondrial Ca2� uptake.13 Liver cells also respond tochanges in extracellular calcium levels when serumcalcium levels rise or fall above the normal range, withthe result that some liver functions such as bile secre-tion and metabolic activity may be compromised.14

To the best of our knowledge, Celsior offers safety andeffectiveness very similar to those of University of Wis-consin (UW) solution for cold storage preservation ofliver grafts, as confirmed in clinical and experimentalstudies.15 In our study, we performed experiments us-ing Celsior for cold storage preservation: this solutioncontains a Na/K ratio similar to that of the University ofWisconsin–gluconate (UW-G) solution used for the hy-pothermic MP liver preservation. We decided to performMP using Krebs-Heinseleit (KH) modified solution for 3main reasons: (1) KH has a Na/K ratio, buffering capac-ity, and energy substances very similar to those ofUW-G; (2) calcium is present in KH but absent in UW-G,and calcium deprivation has been shown to induce cho-lestasis at least in part because of a disturbance in theosmotic equilibrium16; and (3) UW-G contains hydroxy-ethyl starch, which may cause deficits during MP pres-ervation because of its high viscosity,17 especially withthe high flow used during MP. In our solution, n-acetyl-cysteine (NAC) replaces glutathione because of its anti-oxidant and microcirculatory relaxant properties.18 Theintention of our article is not to question the suitabilityof UW-G solution for organ preservation by MP but toraise the awareness that there are alternatives that mayhave a better performance profile, particularly when anew experimental setting is used in which the preser-

vation temperature is modified. Furthermore, althoughthere are some crucial differences between the Celsiorand KH solutions, they have been used to compare the2 preservation methods, namely, cold storage andMP.19

On the basis of these considerations, the goals of thisstudy are (1) to investigate the relationship between theliver temperature employed during MP and reperfusiondamage, (2) to compare hepatic damage induced by MPwith that induced by conventional cold storage, and (3)to examine the role of Ca2� levels in the MP solution.

MATERIALS AND METHODS

Animals and Surgery

Male Wistar rats (Harlan-Nossan, Italy), weighing 250-300 g, were allowed free access to water and food untilthe beginning of all experiments. The use and care ofanimals in this experimental study were approved bythe Italian Ministry of Health and by the UniversityCommission for Animal Care. Rats were anesthetizedwith sodium pentobarbital (40 mg/kg intraperitoneally)and received 250 units of heparin via the inferior venacava. The bile duct was cannulated with 50 gauge poly-ethylene tubing, and an intravenous catheter (16-gauge) was inserted into the portal vein. The liver waswashed out with an oxygenated KH medium containing2.5 mM CaCl2 (KH), 20 mM 4-(2-hydroxyethyl)-1-piper-azine ethanesulfonic acid, 5 mM glucose, and 5 mMNAC (pH 7.4; 4 mL/minute/g of liver) and removed.20

The isolated perfused liver represents a standard modelfor monitoring preservation damage in rat livers21 evenwithout the reperfusion of the hepatic artery.22 Sam-ples of control livers were obtained immediately afterwashout.

MP

The liver was placed in an organ chamber and con-nected to recirculating standard perfusion equipmentset up for 6 hours containing 200 mL of KH medium ata fixed temperature (4°C to 37°C). KH solution wasrecirculated by a roller pump (Gilson Minipuls 3), oxy-genated (600-650 mm Hg of oxygen pressure), andmaintained at a fixed temperature by a heat exchanger(Julabo F12). The perfusate ran freely via the suprahe-patic caval vein into the organ chamber and was imme-diately recirculated into the reservoir by the rollerpump. Air emboli were removed from the system by abubble trap; portal venous pressure was monitored by awater column connected to the portal vein inflow cath-eter. Experiments using KH with 1.25 mM CaCl2 (KH–1.25 mM CaCl2) or KH with 0.25 mM CaCl2 (KH–0.25mM CaCl2) were also performed. To evaluate the role ofmitochondrial Ca2� accumulation during MP liver pres-ervation, ruthenium red (RR; 10�M),23 an inhibitor ofmitochondrial Ca2� uniporter, was added to KH–2.5mM CaCl2.

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Cold Storage

After washout with KH, the livers were flushed in situwith ice-cold Celsior solution (Pasteur Merieux Serumset Vaccins, Lyon, France) for 2 minutes, maintained inthis ice-cold solution, and placed in plastic containerssurrounded by ice for 6 hours.

Normothermic Reperfusion

Reperfusion with KH (2 hours at 37°C) was performedunder the same conditions in MP-preserved and coldstorage–preserved livers. The initial flow rate was 1 mL/minute/g, which was gradually increased to 4 mL/minute/g over 5 minutes. The perfusion pressure (con-trolled by gravity), temperature, and pH of the perfusatewere constantly monitored, and the liver was placedinto a thermostat-controlled chamber.

Assays

Hepatocyte viability was assessed by the release of lac-tate dehydrogenase (LDH) into the effluent perfusateduring the experiments.24 Aspartate aminotransferase(AST) and gamma-glutamyl transpeptidase (�GT) levelsin the perfusate or in the bile were determined with aHitachi 747 automated analyzer (Roche/Hitachi, Indi-anapolis, IN). Bile was collected in a graduated tubeduring the reperfusion period to estimate the rate of bileproduction (�L/g of liver tissue). Tissue ATP was mea-sured by the luciferin-luciferase method with the ATPBioluminescence Assay Kit CLS II (Roche MolecularBiochemicals, Milan, Italy). Protein was assayed by themethod of Lowry et al.,25 with bovine serum albuminused as the standard.

Morphology

Samples of the liver were quickly removed, and smallfragments were fixed by immersion in 2.5% glutaralde-hyde in 0.13 M Milloning buffer (pH 7.2-7.4) at 4°C for4 hours, rinsed, postfixed with 1% osmium tetroxide at4°C for 2 hours, washed, dehydrated through gradedconcentrations of alcohol, and embedded in Epon.Semithin sections (1 �m thick) were stained with 1%

toluidine blue and observed with a Zeiss Axioscop Pluslight microscope. Ultrathin sections were stained withuranyl acetate for 7 minutes and lead citrate for 2 min-utes, coated with carbon, and observed with a Zeiss EM900 electron microscope operating at 80 kV.

Data Analysis

Data are presented as the mean � standard error from4 to 6 independent experiments. Statistical analysis formultiple comparisons was performed by a 1-way anal-ysis of variance and Scheffe post hoc test and withBonferroni’s corrections. P � 0.05 was the criterion ofsignificance.

RESULTS

Role of Temperature

In the first part of this study, the relationship betweenthe liver temperature employed during MP and reper-fusion damage was investigated by the performance ofexperiments at 4°C, 10°C, 20°C, 25°C, 30°C, and 37°C.MP at 37°C caused marked AST and LDH release (Table1); the same was obtained during reperfusion (Table 2).By contrast, MP temperature lowering induced a signif-icant and temperature-dependent decrease in liverdamage, about 10 times more than that of MP at 37°C(Table 1). Enzyme release during reperfusion showed adecrease in liver damage associated with the tempera-ture used during MP preservation: perfusate activitiesof AST and LDH were significantly decreased in the 4°Cand 10°C group in comparison with all other groups(Table 2).

Bile flow during MP preservation was strongly depen-dent on the increase in the temperature used up to 30°C,whereas at 37°C, it reached a peak after 3 hours of per-fusion (data not shown). On the contrary, during reperfu-sion, bile production was proportional to the lowering ofthe temperature employed in MP preservation. MP at 4°Cand 10°C showed higher bile flow in comparison with allother temperatures used (Table 2). Biliary complicationsafter orthotopic liver transplantation remain the Achilles’heel of this operation,25-27 and bile analysis after coldischemia represents a tool for assessing the integrity of

TABLE 1. Hepatocellular Injury and Liver Function After 6 Hours of MP at Different Temperatures

Temperature (°C)

Perfusate Bile

AST

(mU/mL)

LDH

(mU/minute/g)

Production

(�L)

�GT

(mU/mL)

LDH

(mU/mL)

37 168.4 � 22 170 � 39 88 � 39 53 � 9 6.4 � 230 54.7 � 7.5 14 � 2.1 111 � 24 35 � 6 5.1 � 425 38.3 � 2.2 10 � 0.8 90 � 11 32 � 5 4.3 � 220 14.8 � 1.6 5.9 � 1.1 42 � 3.4 25 � 8 2.8 � 1.210 11.3 � 0.9* 3.8 � 1.8* 9 � 1.2* 42 � 9* 5.5 � 1.9*4 10.8 � 1.2* 4.1 � 1.3* 8 � 1.7* 45 � 5* 6.1 � 2.1*

NOTE: Each point represents the mean � standard error of 5 different experiments.*P � 0.05 versus MP at 20°C.

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biliary epithelial cells after cold ischemia-reperfusion(I/R) of rat livers.28 �GT and LDH biliary concentrationswere used to quantify bile duct injury during reperfusionafter MP preservation: higher biliary enzymes were ob-served in livers preserved by MP at 4°C and 10°C versusMP at 20°C (Table 2). In particular, �GT was 80 � 9, 78 �4, and 30 � 7 mU/mL (P � 0.05) in livers preserved byMP at 4°C, 10°C, and 20°C, respectively. MP at 4°C and10°C induced a bile enzyme increase in comparisonwith MP at 20°C.

Role of Ca2�

In the second part of this study, we investigated the roleof Ca2� levels in the perfusate medium used for MPpreservation. We employed the KH solution for MP, asolution usually used in the isolated perfused modelcontaining 5 mM glucose.20 On the basis of the resultsobtained in the first part of this study, we performedexperiments at 20°C, a temperature that protected bothhepatocytes and cholangiocytes (Table 2); we used aperfusate containing different Ca2� concentrations asprevious reports suggested that calcium addition maybe one agent for improving liver preservation: the addi-tion of 1.5 mM calcium to the UW solution suppressedthe incidence of damage, which appeared to be similarto damage in the unpreserved liver.29 When KH with1.25 or 0.25 mM CaCl2 was used for MP at 20°C, verylow levels of enzyme release were observed for 6 hoursof MP (AST: 8.9 � 1.5 and 10.1 � 0.8 mU/mL, respec-tively); LDH release started after 1 hour of MP andremained unchanged during the remaining 5 hours(Fig. 1A). This damage was not significantly differentfrom that obtained through MP at 4°C and 10°C (AST:10.8 � 1.2 and 11.3 � 10.9 mU/mL, respectively, P �0.05). Significantly lower hepatic damage was alsopresent at the end of the reperfusion period after MP at20°C using KH with 1.25 or 0.25 mM CaCl2 versusKH–2.5 mM CaCl2 (Fig. 1B). The hepatic injury did notdiffer from that observed in organs preserved by MP at4°C (AST: 20.5 � 3 mU/mL with KH–1.25 mM CaCl2and 17.9 � 2 mU/mL with KH–0.25 mM CaCl2 versus21 � 2.9 mU/mL for MP at 4°C; P � 0.05). Furthermore,

we compared liver damage during reperfusion in MP-preserved and cold storage–preserved livers: AST re-lease after cold storage preservation increased to 55%and 62% of that observed with MP at 20°C using KHwith 1.25 or 0.25 mM CaCl2, respectively (Fig. 1B). Bileproduction during MP at 20°C remained unchangedwith different levels of CaCl2 in the perfusate (Fig. 2A).Conversely, during reperfusion, the bile flow decreasedin a significant manner in liver preserved by MP at 20°with KH–0.25 mM CaCl2 compared with cold storageand MP at 20°C with KH–1.25 mM CaCl2 or KH–2.5 mMCaCl2 (Fig. 2A). Biliary �GT and LDH levels increasedduring the reperfusion period following cold storage butremained low with MP at 20°C under all the experimen-tal conditions studied (Fig. 2B).

RR has a high affinity for the mitochondrial calciumuniporter, a gated ion channel that can be completelyinhibited by micromolar concentrations of this drug.23 Inthis study, protection by RR (10 �M) was observed at theend of reperfusion using KH–2.5 mM CaCl2: liver damagewas similar to that observed with KH–1.25 mM CaCl2 orKH–0.25 mM CaCl2 during MP preservation (Fig. 1).

ATP Levels in MP Preservation

The energy status of hepatic biopsies at the end of thereperfusion period showed that ATP levels were signif-icantly higher in livers preserved by MP at 20°C withKH–0.25 mM CaCl2 or KH–1.25 mM CaCl2 comparedwith cold storage preservation or MP at 20°C with KH–2.5 mM CaCl2 (Fig. 3). RR added to KH–2.5 mM CaCl2restored, at least in part, the ATP levels (Fig. 3).

Morphological Changes

In livers submitted to cold storage, analysis of semi-thin sections revealed edematous portal tracts, hepato-cytes with swollen mitochondria and reticulum, dilatedsinusoids, and protein loss into the lumen of veins andsinusoids (Fig. 4A). By contrast, in liver submitted toMP (KH–1.25 mM Ca2�), tissue morphology was muchbetter preserved (Fig. 4B). Observation under the elec-tron microscope revealed a lobular zone–dependent cell

TABLE 2. Hepatocellular Injury and Liver Function at the End of Reperfusion After MP Preservation

at Different Temperatures

Temperature (°C)

Perfusate Bile

AST

(mU/mL)

LDH

(mU/minute/g)

Production

(�L)

�GT

(mU/mL)

LDH

(mU/mL)

37 2212 � 244 499 � 59 4 � 1 155 � 39 24 � 530 250 � 117 62 � 14 19 � 6 114 � 19 16 � 425 51 � 2.8 16 � 2.1 39 � 4 82 � 12 5.3 � 220 42 � 3.5 12 � 0.8 45 � 4 30 � 8 4.2 � 1.210 23 � 3.1* 6.0 � 2* 58 � 6* 78 � 11* 6.5 � 1.9*4 21 � 2.9* 5.8 � 1.8* 55 � 4* 80 � 9* 6.9 � 1.3*

NOTE: Each point represents the mean � standard error of 5 different experiments.*P � 0.05 versus MP at 20°C.

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response. We can summarize morphological changes inrelation to cold storage and MP with KH–1.25 mMCaCl2.

Cold Storage Followed by Reperfusion

In the periportal region, the connective tissue wasedematous (not shown), hepatocytes contained largeglycogen lakes associated with lipid droplets, and mi-tochondria were swollen (Fig. 5A). Several apoptoticKupffer cells were observed; collagen deposits werepresent in Disse’s space, and the lumen of sinusoidswas crowded with cell debris (not shown). In the cen-trolobular region, hepatocytes did not contain glycogenor lipid droplets; mitochondria were swollen, and sev-eral residual bodies were present (Fig. 5C). A fibroticreaction was very marked in Disse’s spaces (Fig. 6B,C).

MP with KH–1.25 mM Calcium Followed byReperfusion

In the portal region, edema was absent, and hepato-cytes were very well preserved. They contained abun-

dant glycogen but almost no lipid droplets, and themorphology of mitochondria was well preserved (Fig.5B). The lumen of sinusoids was pervious, and bileducts appeared to be undamaged (Fig. 6A). In the cen-trolobular region, hepatocytes still contained glycogen;their mitochondria were well preserved, but the cyto-plasm contained a high number of residual bodies (Fig.5D). Cell debris, much less abundant than with coldstorage, was observed in the sinusoid lumen, especiallyin the centrolobular zone (not shown). A few apoptoticKupffer cells were seen (not shown).

DISCUSSION

The presented results indicate that MP at 20°C with KHcontaining 1.25 mM Ca2� reduces liver susceptibilityassociated with organ preservation in comparison withconventional cold storage. We may note the crucial rolethat Ca2� plays in the reduction of liver damage duringpreservation by MP at 20°C, exhibiting a marked reduc-

Figure 1. Effect of Ca2� levelsand RR on liver injury duringpreservation and reperfusion.Livers were preserved by MPwith an oxygenated KH mediumcontaining 0.25, 1.25, or 2.5 mMCaCl2 at 20°C or by cold storageat 4°C for 6 hours. RR (10�M)was added to KH–2.5 mM CaCl2during MP preservation. Thereperfusion period with KH (2hours at 37°C) was performedunder the same conditions inMP-preserved and cold storage–preserved livers. (A) AST andLDH release during MP washigher with KH–2.5 mM CaCl2than with KH–0.25 mM CaCl2,KH–1.25 mM CaCl2, or KH–2.5mM CaCl2 � RR. (B) AST and LDHrelease during reperfusion washigher with KH–2.5 mM CaCl2and cold storage than with KH–0.25 mM CaCl2, KH–1.25 mMCaCl2, or KH–2.5 mM CaCl2 �RR. These are the mean resultsof 4 to 6 different experiments �the standard error of the mean.*P < 0.05 versus MP with KH–0.25 mM CaCl2, KH–1.25 mMCaCl2, or KH–2.5 mM CaCl2 �RR. **P < 0.05 versus MP withKH–0.25 mM, KH–1.25 mMCaCl2, or KH–2.5 mM CaCl2 �RR.

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Figure 2. Effect of Ca2� levels inthe MP perfusate on bile flow andenzyme release during MP pres-ervation and reperfusion. Liverswere preserved by MP with an ox-ygenated KH medium contain-ing 0.25, 1.25, or 2.5 mM CaCl2at 20°C or by cold storage at 4°Cfor 6 hours. The reperfusion pe-riod with KH (2 hours at 37°C)was performed under the sameconditions in both MP-preservedand cold storage–preserved liv-ers. (A) Bile flow in MP preserva-tion and in the reperfusion pe-riod is shown. Bile flow duringreperfusion after MP withKH–0.25 mM CaCl2 was lowerthan with the KH medium con-taining 1.25 or 2.5 mM CaCl2 andcold storage. *P < 0.05 versuscold storage and MP with KH–2.5mM CaCl2 and KH–1.25 mMCaCl2. (B) At the end of reperfu-sion, cold storage inducedhigher release of �GT and LDHinto bile than MP. These are themean results of 4 to 6 differentexperiments � the standard er-ror of the mean. *P < 0.05 versusMP with KH–0.25 mM CaCl2,KH–1.25 mM CaCl2, or KH–2.5mM CaCl2.

Figure 3. Effect of Ca2� levels on ATP concen-trations at the end of reperfusion. Livers werepreserved by MP with an oxygenated KH mediumcontaining 0.25, 1.25, or 2.5 mM CaCl2 at 20°Cor by cold storage at 4°C for 6 hours. RR wasadded to KH–2.5 mM Ca2� during MP preserva-tion. The reperfusion period with KH (2 hours at37°C) was performed under the same conditionsin MP-preserved and cold storage–preserved liv-ers. ATP levels with the KH medium containing0.25 or 1.25 mM CaCl2 were higher than with 2.5mM CaCl2 and cold storage (*P < 0.05). These arethe mean results of 4 to 6 different experi-ments � the standard error of the mean.

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tion in necrosis usually exacerbated by preservation at4°C.

Most published works have reported data about amodel of MP liver preservation performed under hypo-thermic conditions: MP is usually performed at 4°C. Therole of temperature has not so far been considered, andconsequently, the optimal temperature during MP needs

to be defined. A strong correlation between the liver tem-perature employed during MP and reperfusion damagehas been demonstrated in our study for the first time: MPat 20°C was found to be the optimal temperature forpreserving both hepatocytes and cholangiocytes, as re-vealed by limited enzyme release, ATP levels, and mor-phology. In agreement with the study of Fujita et al.,30

oxygen consumption in the liver also depends on the tem-perature used: the relation between the hepatic oxygenrequirement and temperature is logarithmic, and oxygenconsumption decreases with decreasing temperature. At20°C, the oxygen requirement is markedly reduced com-pared with that at 37°C (0.7 and 2.5 nmol/minute/g,respectively). It may be that the hepatic oxygen requestduring MP at 20°C is nearly equivalent to the oxygensupply in our MP model, as highlighted by cell integrity,minimum enzyme release, and improved liver functionand morphology during reperfusion. Studies on the role oftemperature versus oxidative stress during hepatic reper-fusion have demonstrated that hypothermia at 15°C com-pletely inhibits oxidant-induced tissue damage and thatthe protection is already present after the organ coolsdown to 32°C to 26°C.31 Furthermore, the analysis of theinfluence of different ischemia temperatures in a mouseexperimental model of hepatic I/R injury demonstratedprotective effects even of mild hypothermia at 26°C.32 Wemay posit that liver preservation by MP at 20°C instead ofa hypothermic temperature reduces vasoconstriction ofhepatic vasculature and increases flow resistance be-cause no significant increase in perfusion pressure dur-ing MP or reperfusion occurred (data not shown). Mor-

Figure 4. Light microscopy of semithin sections stained withtoluidine blue of portal areas from livers submitted (A) to coldstorage followed by reperfusion or (B) to MP at 20°C with KHcontaining 1.25 mM Ca2� followed by reperfusion. (A) Intracel-lular edema and loss of protein in the lumen of the vein can beobserved in the livers submitted to cold storage, whereas (B)tissue morphology was better preserved when the livers weresubmitted to MP. The scale bar is 50 �m. Abbreviations: D, bileduct; P, portal vein.

Figure 5. Electron micrographs of hepatocytes of livers sub-mitted (A,C) to cold storage and reperfusion or (B,D) to MP at20°C with KH containing 1.25 mM Ca2� and reperfusion. (A,B)Portal hepatocytes and (C,D) centrolobular hepatocytes areillustrated. The arrows indicate residual bodies. The scale baris 1.1 �m. Abbreviations: G, glycogen; L, lipid droplets.

Figure 6. Electron micrographs of (A) a well-preserved portalregion in the liver submitted to MP at 20°C with KH 1.25 mMCa2� and reperfusion and (B,C) fibrosis in a centrolobular areaof livers submitted to cold storage and reperfusion. The scalebars are (A,B) 2.5 and (C) 1.7 �m. Abbreviations: C, collagenfibers; D, bile duct; f, fibroblast; m, (myo)fibroblast; M, mastcell.

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phology revealed pervious sinusoids in most lobuli. TheNAC addition to the perfusate could also be involved inthe control of pressure for its microcirculatory relaxantproperties.

Preservation injury to intrahepatic ducts is consid-ered a weakness of current liver transplantation sur-gery because of its frequency, approximately 10% to50%,33,34 and its potentially lethal effects on the sur-vival of the graft and patients.35 For efficient liver pres-ervation, both hepatocytes and cholangiocytes need tobe protected, and this is what occurs with MP at 20°Cwith KH–1.25 mM CaCl2. MP at 4°C and 10°C providedparenchymal cell protection but marked damage tocholangiocytes, as revealed by an analysis of biliaryenzyme release. The ability to synthesize bile constitu-ents and secrete them across the apical membraneswas probably well maintained in hepatocytes of liverssubmitted to MP at 4°C and 10°C. Lower enzyme release(2-fold) was observed into the perfusate during MP at4°C and 10°C with respect to MP at 20°C. Bile flowincreased with lower temperatures, but at 4°C and10°C, an increase of �GT and LDH was observed. Theelevation of enzymes in the bile confirmed the increasedbile duct injury after liver cold preservation. Our dataare in keeping with the results of Accatino et al.36: nocorrelation exists between bile flow and �GT levels inbile. We decided to use KH–1.25 mM CaCl2 rather thanKH–0.25 mM CaCl2 because bile flow occurs only whenan adequate Ca2� concentration exists in the perfusionmedium.16 Some reports provide evidence of a role forCa2� in the modulation of bile flow, confirming theresults of our work: bile flow is attenuated when aCa2�-free perfusate medium is employed37 or whenexcessive ethylene glycol tetraacetic acid has beenadded.38 Calcium deprivation has been shown to in-duce cholestasis due, at least in part, to a disturbancein the osmotic equilibrium possibly caused by impair-ment of an ion transport system involved in hepatocel-lular volume control.16 The use of KH–1.25 mM CaCl2was also supported by the role of extracellular Ca2� in

hepatic bile formation39 and in the active contraction ofbile canaliculi.40 Morphological analysis confirms thatbile ducts are undamaged in liver preserved by MP with1.25 mM CaCl2.

Recently, the addition of 2 mM Ca2� to the modifiedUW solution during liver MP demonstrated significantlylower oxidative stress and improved liver function com-pared with Ca2�-free UW perfusion.41 Furthermore, itwas recently reported that a reduction in the extracel-lular Ca2� concentration from 0.2 to 0.05 mM inhibitedthe rate of bile flow by approximately 85%.42 Our re-sults from MP with KH–0.25 mM Ca2� confirm thatvery low levels of Ca2� in the perfusate are associatedwith significant bile flow reduction. Conversely, it isnoteworthy that a high intracellular Ca2� concentra-tion has been found to play an important role in hepaticI/R injury.43 In particular, cold storage affects mito-chondrial Ca2� handling, especially when it is chal-lenged by a high extramitochondrial Ca2� concentra-tion.23 After cold ischemic storage of the liver,activation of mitochondrial Ca2� uptake occurs at theexpense of the mitochondrial transmembrane gradient,which is maintained by reversal of F0F1-type proton-ATPase synthase. In these circumstances, the mito-chondrial Ca2� overload is associated with cell ATPdepletion and mitochondrial permeability transitionpore formation.44 The higher ATP levels observed in theliver preserved by MP at 20°C with KH–1.25 may beexplained by a limited mitochondrial Ca2� overload.Our data suggest that 1.25 mM CaCl2 in the perfusatemedium is a good compromise for liver preservationavoiding cholestasis and Ca2�-dependent intracellularpathways. We can speculate that a perfusion mediumcontaining 1.25 mM Ca2� protects mitochondria andmaintains better hepatic ATP levels. Our hypothesishas been confirmed by the experiments in which RRwas added to the MP solution: this drug protected liverpreserved with KH–2.5 mM CaCl2, suggesting a role formitochondria in the protection of MP medium contain-ing 1.25 mM CaCl2. Inhibition of mitochondrial Ca2�

Figure 7. RR protects mitochon-dria from Ca2� uptake. When MPof livers is performed with KH–2.5 mM Ca2�, (A) the ion entersinto the mitochondria via theuniporter at the expense of thetransmembrane potential (m��);(B) hydrolysis of ATP by reversedF0F1-type synthase restores thepotential, and with KH–1.25 mMCa2�, a small amount of ion en-ters the mitochondria; and (C)RR inhibits uniporter-dependentmitochondrial calcium uptakewith KH–2.5 mM Ca2�, prevent-ing ATP consumption by preser-vation of the mitochondrialtransmembrane gradient.

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uniporter with RR protects mitochondria from Ca2�

overload at a high Ca2� concentration and attenuatesthe effect of either cold or warm I/R injury in the liv-er.23,45 It has been established that rapid uptake ofCa2� by mitochondria from a wide range of sources ismediated by a uniporter that permits transport of theion down its electrochemical gradient. Energized mito-chondria must expend a significant amount of energy totransport Ca2� against its electrochemical gradientfrom the matrix space to the external space. Our dataindicate that RR, a gated ion channel, protect mito-chondria from Ca2� uptake, allowing a saving of ATP bypreserving the mitochondrial transmembrane gradient,as depicted in Fig. 7.

The relevant findings in this study are a demonstra-tion that a correlation exists between the temperatureused in MP liver preservation and reperfusion damageand that calcium levels in the perfusate play a crucialrole in the control of this injury. MP at 20°C with KH–1.25 mM CaCl2 afforded better protection of liveragainst reperfusion damage than conventional coldstorage. Mitochondria appear to play an important rolein liver protection by MP, but further investigationsneed to be performed to clarify this point. This prelim-inary investigation may open up new routes for improv-ing organ preservation and suggest in particular thatsubnormothermic liver MP may represent a new strat-egy against preservation damage. The addition of anoptimal calcium concentration to the perfusate may beone agent for improving liver function.

ACKNOWLEDGMENT

We thank Mr. Gaetano Viani for his skillful technicalassistance. We also thank Professor Anthony Baldry forrevising the English.

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