Protective strategies against ischemic injury of the liver

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GASTROENTEROLOGY 2003;125:917–936

PECIAL REPORTS AND REVIEWS

rotective Strategies Against Ischemic Injury of the Liver

AZIA SELZNER, HANNES RUDIGER, ROLF GRAF, and PIERRE–ALAIN CLAVIEN

his article summarizes strategies to protect the liverrom injuries caused by ischemia and reperfusion. Threeifferent sections (i.e., surgical and pharmacologic strat-gies and gene therapy) present approaches to enhancehe survival and viability of the liver in various surgicalrocedures including liver transplantation. The first sec-ion reviews approaches using surgical interventionsuch as ischemic preconditioning and intermittentlamping. Their protective effects are discussed withespect to the mechanism of injury. In the second sec-ion, pharmacologic agents targeting microcirculation,xidative stress, proteases, and inflammation are de-cribed. Mechanisms of injury and their suppression bywide variety of drugs are discussed. The third section

ocuses on gene therapy. Potential target genes haveeen identified (e.g., superoxide dismutase or hemexygenase). Animal experiments in which the liver injury

s reduced successfully may pave the way to novel strat-gies applied to different liver diseases in humans.

ew areas in medicine have enjoyed similar success asliver transplantation. As a result, the imbalance be-

ween organs available for transplantation and the num-er of patients awaiting an organ has grown dramaticallyver the past decade, triggering interest to maximize andptimize the use of potential organs. For example, mar-inal organs (i.e., organs not used previously or expectedo be associated with increased risk for malfunction) andartial liver transplantation such as living-related andplit-liver transplantations are used increasingly in mostransplant centers.1,2 A common issue inherent to alltrategies is the need to preserve the graft from the timef harvesting until implantation.3 From cooling of theraft, initiated in the 1950s, and the introduction of theniversity of Wisconsin (UW) cold-storage solution for

old preservation in the mid-1980s,4 many experimentaltudies have suggested novel protective strategies, al-hough very few have yet reached clinical practice. Sim-larly, the volume of liver surgery as part of the trans-lant process (e.g., living-related liver transplantation) oror resection of tumors has increased dramatically overast years worldwide,5 and strategies to minimize the

egative effects of ischemia are now in the forefront oflinical and experimental studies related to liver resec-ion. This article reviews established and promising pro-ective strategies against ischemic injury of the liver.

Should We Differentiate DifferentTypes of Ischemic Injury of theLiver?The liver can be subjected to 3 forms of ischemia,

amely cold (or hypothermic), warm (or normothermic),nd rewarming.3 Cold ischemia occurs almost exclusivelyn the transplant setting where it is applied intentionallyo reduce metabolic activities of the graft while the organwaits implantation. Warm ischemia occurs in a varietyf situations including transplantation, trauma, shock,nd liver surgery, when hepatic inflow occlusion (Pringleaneuver) or inflow and outflow (total vascular exclu-

ion) are induced to minimize blood loss while dividinghe liver parenchyma. Rewarming ischemia typically oc-urs during manipulation of the graft (e.g., ex situ splitiver preparation) or during the period of implantation ofhe graft when the cold liver is subjected to room or bodyemperature while performing the vascular reconstruc-ion. Of note, injury to the liver cells after any type ofschemia is detected mainly after reperfusion when oxy-en supply and blood elements are restored. Morphologictudies in various animal models have shown majorifferences in the patterns of cold and warm injury. Inhe 1980s, it was shown that cold ischemia specificallyaused injury to the sinusoidal endothelial cell (SEC),6–8

finding supported by many subsequent studies.9–12 TheEC detached, lost cytoplasmic processes, becameounded as a result of alteration of the extracellularatrix and cytoskeleton, and sloughed into the sinusoi-

Abbreviations used in this paper: Hsp, heat shock protein; IL, inter-eukin; MMP, matrix metalloprotease; OH, hydroxyl radical; SEC, sinu-oidal endothelial cell; TNF-�, tumor necrosis factor �; UW solution,niversity of Wisconsin cold-storage solution.

© 2003 by the American Gastroenterological Association0016-5085/03/$30.00

doi:10.1016/S0016-5085(03)01048-5

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dal lumen (Figure 1).7,9,13 Despite these structuralchanges, most SECs remain alive during the period ofischemia14,15 but rapidly die on reperfusion. Disruptionof the endothelial wall leads to leukocyte16–20 and plate-let adhesion,21,22 which induces microcirculatory distur-bances.23 The degree of endothelial cell detachment has

been shown to correlate with the duration of cold isch-emia in numerous experimental models.9,13,19 The mor-phologic changes typically identified in the endothelialcells result from active processes involving the cytoskel-eton and extracellular matrix.24,25 We have describedbiologic similarities between the injury of the SECsexposed to cold and the early stages of angiogenesis asseen in wound healing and tumor growth.26 In thesecases, the endothelial cells undergo apoptosis on reper-fusion.10

Adhesion of platelets to the sinusoid lining inducesSEC apoptosis on reperfusion of the cold ischemic liver.22

Platelets are a rich source of transforming growth factor�27 and calpains.28 Nitric oxide production by plateletsin combination with oxygen free radical synthesis onreoxygenation of the ischemic liver can lead to the for-mation of perioxynitrite, a highly reactive inducer ofapoptosis in endothelial cells.29 Finally, models using theisolated perfused rat liver revealed that leukocytes andplatelets synergistically exacerbate SEC injury by induc-tion of apoptosis and that Kupffer cells are involved inthe mechanism of injury mediated by these cells.30

In contrast to cold ischemia, warm ischemia is toler-ated poorly and rapidly leads to the death of hepato-cytes.31,32 This severe injury of the hepatocytes probablyis preceded by massive death of endothelial cells33 (Fig-ures 1 and 2). The role of Kupffer cells, the residenthepatic macrophages, and adherent leukocytes and plate-lets remains an area of active investigation in the warmischemic liver.34 On reperfusion, Kupffer cells are acti-vated.30,35,36 This is evidenced by structural changes,35

formation of oxygen free radicals,17,37 increased phago-cytosis, and release of lysosomal enzymes10,38 and variouscytokines including tumor necrosis factor � (TNF-�)38,39

(Figure 3). Further binding of these cytokines to theirrespective receptor or release of oxygen free radicalsduring early stages of reperfusion initiates the complexapoptotic machinery leading to the death of hepato-cytes.38 Stress-activated protein kinase, especially c-jun

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Figure 1. Representative transmission electron micrographs of ratlivers preserved in cold Euro-Collins solution for 16 hours of preser-vation (nonviable condition) and/or reperfused for 1 hour in the IPRLmodel. (A) Cold-preserved liver after 16 hours of cold ischemia. Notethe typical cold preservation injury with rounding and detachment ofendothelial cells and disruption of the sinusoidal wall. (B) Liver pre-served for 16 hours and reperfused for 1 hour. An apoptotic endothe-lial cell is shown with typical shrinkage and chromatine condensationin the nucleus. Note the presence of intact mitochondria and numer-ous vacuoles (arrow). (C) In the same group are numerous activatedKupffer cells with phagolysosomes containing apoptotic bodies. S,sinusoid; EC, endothelial cell; K, Kupffer cell; L, lymphocyte; H, hepa-tocyte; R, red blood cell; A, apoptotic bodies. Reprinted with permis-sion of Clavien et al.3 and Gao et al.10

918 SELZNER ET AL. GASTROENTEROLOGY Vol. 125, No. 3

N terminal kinase, are activated by extracellular stimuliduring hypoxia reoxygenation, leading to nuclear tran-scription factor activation and to the onset of hepatocyteapoptosis.40,41

After reperfusion, leukocytes rapidly adhere to thedenuded sinusoids and contribute significantly to theinjury.16,8-20,42 Both TNF-� and interleukin 1 (IL-1),released by activated Kupffer cells, can up-regulateCD11b expression on leukocytes and recruit these cellsinto sinusoids.43 The mechanism of injury involves therelease of reactive oxygen intermediates by reduced nic-otinamide adenosine dinucleotide phosphate–dependentoxidase systems expressed on the membrane surface ofneutrophils. Other potential substances released by neu-trophils include various proteases and hypochloric acid.44

There is a growing body of evidence suggesting that hostT cells also participate in hepatic ischemic injury. Forexample, both cyclosporine A and Fk506, which arepotent T-cell–deactivating agents, may decrease reper-fusion injury after transplantation or warm ischemia.45,46

TNF-� and IL-1 also can recruit and activate CD4� Tlymphocytes in the liver during the early phase of reper-fusion,47 which auto-amplify Kupffer cell activation andneutrophil recruitment into the liver. These phenomenaare mediated through the release of several factors byCD4� T lymphocytes, such as granulocyte colony stim-ulating factor and interferon �.47,48 The interaction be-tween CD 154 expressed on mature activated CD4� Tlymphocytes and CD40 on antigen-presenting cells,

causes a T-cell–mediated type of injury in a model ofwarm ischemia in mice.49

Leukocyte recruitment into sinusoids during the earlyphase of reperfusion also is mediated through activationof the complement cascade. The complement chains ac-tivated rapidly by cellular proteins released during reper-fusion up-regulate macrophage antigene 1 expression onneutrophils and their recruitment into sinusoids.50 Ad-ditionally, complement components can directly causecell injury by assemblage and deposition on mem-branes.51

The impact of rewarming on the structural integrity ofthe liver and the mechanism of this type of injury isunderstood poorly. It probably reflects a combination ofcold and warm injury.

How Can We Protect the LiverAgainst Ischemic Injury?Many protective strategies have been proposed,

which can be classified in different ways. For example,from a practical perspective, protective strategies can bedivided into 3 different categories: (1) surgical interven-tions, (2) the use of pharmacologic agents, and (3) gene

Figure 2. Electron micrograph representing warm injury in the mouseliver. Endothelial cell swelling accompanied by hepatocyte necrosis(nH) is observed. Accumulation of polymorphonuclear leukocytes (PMN)and platelets (PLTs) are present in the sinusoids. R, red blood cell.

Figure 3. Mechanisms of warm ischemic injury. Major pathways in-clude TNF-�–mediated apoptosis, dysregulation of ion distribution,and the generation of reactive oxygen intermediates (ROI). Intracellularsodium accumulation is caused by a combination of blocking sodiumefflux driven by the Na�/K� adenosine triphosphatase and activationof sodium influx by the Na�/H� exchanger. In the cold, similar factorsfrom the sinusoidal lumen cause injury on SECs. After reperfusion andrewarming, these cells rapidly undergo apoptosis.

September 2003 PROTECTION AGAINST LIVER ISCHEMIA 919

therapy. Another possibility is to present the strategiesbased on the protective mechanisms: (1) strategies aimedat a preemptive induction of tolerance against ischemicinjury, which can be covered by the concept of precon-ditioning; and (2) strategies aimed directly at interferingwith the pathways of injury either by inhibiting delete-rious molecules or enhancing protective pathways. Thiscan be covered by the term direct protection.

In this article, we use the first classification because ithas more practical implications. Protective effects againstcold and warm ischemic injury are discussed for eachstrategy. Although some protective strategies also mayaffect hepatic regeneration, we limit this review on isch-emic and reperfusion injury. For an in-depth review onsome specific aspects of hepatic injury, the reader isreferred to recent reviews.52–55

Surgical StrategiesToday, 2 powerful strategies are in clinical use,

ischemic preconditioning and intermittent clamping(Figure 4). Other protocols that have shown protection inanimal models include preconditioning by hyperther-mia56–61 and application of a portosystemic shunt duringthe hepatic inflow occlusion,23 but these approachesnever made the transition into clinical practice outside ofcase reports.

Ischemic Preconditioning

Ischemic preconditioning consists of a brief pe-riod of ischemia followed by a short interval of reperfu-sion before the actual surgical procedure, with a pro-longed ischemic stress.62 During the surgery, hepatic

inflow is occluded by placing a vascular clamp or a looparound the portal triad (i.e., portal vein, hepatic artery,and bile duct), rendering the whole organ ischemic. Afteran ischemic interval of 10–15 minutes, the clamp isremoved and the liver is reperfused for 10–15 minutesbefore the prolonged ischemic insult (Figure 4). Ourcurrent understanding of the underlying biologic prin-ciple is that cells primed by various kinds of subinjuriousstress trigger defense mechanisms against subsequentlethal injury of the same or different type.54,63,64 Thephenomenon initially was discovered in the myocardiumby Murry et al.65 in 1986. Subsequently, beneficial ef-fects were shown in various tissues including skeletalmuscle,66 brain,67 spinal cord,68 kidney,69 retina,70

lung,71 intestine,72 and liver.73–76 Although the benefitof ischemic preconditioning in the liver already has beensuggested in a clinical pilot study75 and a large random-ized study,77 knowledge of the molecular mechanismsremains vague. Several mediators have been proposed toplay a critical role in the protective pathways includingadenosine,62 nitric oxide,74 oxidative stress, some heatshock proteins (Hsps) (e.g., Hsp 72 and heme oxygenase1/Hsp32),78 and TNF-�.79 In 1990, Colletti et al.39,80

provided evidence suggesting that prolonged ischemicintervals lead to a burst of various cytokines includingTNF-�. Other groups subsequently confirmed this find-ing.79,81,82 TNF-� initiates apoptosis (programmed celldeath) in hepatocytes and SECs.38,82 Peralta et al.79

showed the protective effect of ischemic preconditioningin a rat model of warm ischemia through blockade of Pselectin up-regulation induced by TNF-�.

Several proapoptotic proteins are activated during thereperfusion phase, including the proteases caspase-8 andcaspase-3, and the release of cytochrome c from themitochondria into the cytoplasm. This cascade finallyleads to the destruction of nuclear DNA, resulting in celldeath. However, a controversy emerged over the past yearon whether necrotic or apoptotic cell death accounts forthe severe parenchymal injury observed during reperfu-sion.31,83 Some investigators reported that the over-whelming part of parenchymal injury is caused by mas-sive necrotic alterations.31 In contrast, others showed thatspecific inhibition of apoptosis significantly preventedparenchymal injury and improved animal survival afterprolonged periods of ischemia.11,82,84-87 It is possible thatboth pathways are present after ischemic injury and thatapoptosis and necrosis might overlap after reperfusioninjury. This necroapoptosis theory was developed byLemasters.88 It postulates that a process begins with acommon death signal or toxic stress that culminates ineither cell lysis (necrotic cell death) or programmed

Figure 4. Continuous ischemia is used to prevent bleeding duringliver resection. Three different surgical strategies are shown. Isch-emic periods are drawn in black, and reperfusion is drawn in white.


cellular resorption (apoptosis), depending on other mod-ifying factors such as the decline of cellular adenosinetriphosphate levels or fat content (steatosis).89,90 In thisperspective, it seems possible that the demarcation be-tween apoptosis and necrosis might not be as clear-cut aswas pointed out in numerous publications (Figure 5).

Although the extent of apoptosis is still under debate,it is clear that ischemic preconditioning down-regulatesischemia reperfusion injury: the burst of TNF-� (seeearlier) is inhibited and therefore activation of the apo-ptotic cascade is not observed.75,76 Furthermore, precon-ditioning preserved the morphology of the hepatic pa-renchyma and prevented a rapid increase of generalmarkers of hepatocyte injury such as the serum transam-inase levels.

The protective effects of ischemic preconditioningcould be mediated through various possible mechanisms.Peralta et al.91 identified in a model of warm ischemia aprotective pathway initiated by ischemic precondition-ing that involved the activation of nitric oxide synthaseby adenosine. They found that blocking the specificadenosine receptor A2 prevented the beneficial effect ofischemic preconditioning (see Pharmacologic Strategies).An increase in nitric oxide production was detectedimmediately after hepatic preconditioning. It previouslyhas been reported that nitric oxide, in a relatively narrowtherapeutic window, can inhibit apoptosis by variousmechanisms including the direct inhibition of caspasesby nitrosylation of the active site.92

A second possible mechanism of protection is thatischemic preconditioning confers subinjurious stress tothe liver leading to the development of natural defensemechanisms. Indeed, short intervals of ischemia as ap-plied during ischemic preconditioning induce varioustypes of stress including generation of oxidative stress.Our group recently has shown in a model of warmischemia that a mild burst of oxidative stress generatedduring the process of ischemic preconditioning inducesnatural defense mechanisms against subsequent lethal

injury.93 Similar findings have been reported in coldischemic liver.93,94

In addition to the extracellular mediators, studies inheart and liver indicate that the ischemic precondition-ing process involves activation of intracellular messen-gers such as protein kinase C, adenosine monophos-phate–activated protein kinase, p38 mitogen-activatedprotein kinase, Ik kinase, and signal transducers andactivators of transcription-1.95–98 The downstream con-sequences of these pathways could be cytoprotective byabrogation of cell death pathways (such as activation ofvacuolar adenosine triphosphatases, inhibition of intra-cellular Na accumulation, and cell swelling97,99), stimu-lating antioxidant and other cellular protective mecha-nisms, and by initiating entry into the cell cycle.

In the steatotic liver subjected to warm ischemia, apredominance of necrosis as the primary form of celldeath has been observed.100,101 Intracellular mediators ofapoptosis were decreased in steatotic livers subjected toischemia, indicating a failure to activate the apoptoticcascade. A further indication that apoptosis is not thepredominant mechanism of injury was the lack of pro-tection when caspase inhibitors were used; unlike in thecontrol liver, treatment with these antiapoptotic agentswas not effective in fatty livers.101 Thus, the questionarose whether ischemic preconditioning also may preventnecrotic injury. In the murine steatotic liver, our groupshowed protective effects of ischemic preconditioning byreducing necrosis. These effects were associated withrestoration of high adenosine triphosphate levels afterreperfusion.102 Furthermore, clinical evidence suggeststhat patients suffering from hepatic steatosis (20%–50%steatosis) greatly benefit from ischemic precondition-ing.75 One possible explanation could be the necroapop-totic theory.88 Based on this theory the lack of adenosinetriphosphate and/or other changes in fatty livers mightswitch the type of cell death from apoptosis to necrosis.

The protective effects of ischemic preconditioning onhepatic microcirculation have been studied recently.103,104

Improved microcirculation either might be the result ofor the reason for the preserved parenchymal architec-ture.103 In addition, assessment of the hepatic microcir-culation using intravital microscopy or laser Dopplertechnology is limited to the sinusoids in very closeproximity to the capsule. These areas are supplied bypassive oxygen diffusion via the peritoneum even duringcomplete hepatic inflow occlusion and therefore are notrepresentative for the whole organ. With intravital mi-croscopy, we could show reduced Kupffer cell activationand improved microcirculation in a mouse model of

Figure 5. The necroapoptosis hypothesis proposed by Lemasters.88


warm ischemia and reperfusion (unpublished observa-tions).

Although a short cycle of ischemia and reperfusiontriggers protective mechanisms, it also likewise is asso-ciated with injurious elements105 (e.g., generation ofoxidative stress and nitric oxide). Such detrimental ef-fects of ischemic preconditioning might outweigh in partthe protective effects. From this point of view, nonspe-cific ischemic preconditioning might not be the mosteffective strategy. Further studies should focus on cellu-lar mechanisms of ischemic preconditioning. In a secondstep, identification of pharmacologic agents should beattempted, which specifically interfere with the injuriouseffects of ischemic preconditioning. These agents mightbe used to specifically induce protection and, at the sametime, circumvent the negative effects of the short cycle ofischemia and reperfusion.105

Intermittent Clamping

The first attempts to minimize warm ischemicinjury were undertaken by interrupting long ischemicperiods with multiple short intervals of reperfusion (in-termittent clamping).106 Although the protective mech-anisms of this concept still remain elusive, intermittentclamping currently is used in practice by many centers.It is assumed that the protective mechanisms are similarto those described in ischemic preconditioning, mainlyby reduction of apoptosis.82

In a prospective randomized study, Belghiti et al.107

showed that cycles of short intervals of ischemia (15 min)and reperfusion (5 min) provided a high degree of pro-tection in patients undergoing major liver resection. Wehave compared this protocol with ischemic precondition-ing82 and found that both strategies provide comparableprotection for ischemic intervals of up to 75 minutes. Forlonger ischemic intervals, only intermittent clampingconferred significant protection. However, most resec-tions required inflow occlusion of less than 60 minutes.We therefore concluded that ischemic preconditioning ispreferable for most liver resections because each period ofreperfusion in the intermittent clamping strategy maycause significant bleeding.

Extracorporeal Machine Perfusion Systems

Extracorporeal machine perfusion systems havebeen proposed as a tool to provide superior tissue pres-ervation and viable non–heart beating donor organs. Theaim of such systems is to stop the process of biodegra-dation.108,109 By continuously providing the graft withessential substrates (e.g., glucose, amino acids, nucleo-tides, oxygen) combined with permanent disposal oftoxic metabolites,110 it is expected that organ viability

can be maintained better. The system is based on modelsof isolated liver perfusion that have been used widely tostudy the mechanisms of cold preservation injuries. Al-though this technique has been developed primarily as atool for temporal extracorporeal liver support in patientswith liver failure, it also has a potential application inorgan preservation or resuscitation before transplanta-tion.109 St Peter et al.111 and Imber and St Peter112

showed that warm oxygenated sanguineous machine per-fusion recovers the liver function to a viable level after 24hours of cold preservation whereas simple cold storage(UW solution) for 24 hours renders the liver nonviable.

The perfusion techniques were improved by variousmodifications including oscillating pressure profilesimitating the intra-abdominal conditions113 or simul-taneous dialysis of the recirculating perfusate remov-ing water-soluble toxins and allowing regulation of the pHand electrolyte levels.114,115 Both warm110,111,114–117 andhypothermic118 perfusion systems have been described.For example, using a warm extracorporeal perfusion sys-tem with a porcine liver, Butler et al.116 were able tomaintain the liver in a viable condition for 72 hours.

The introduction of extracorporeal perfusion systemsinto the clinical routine mainly will depend on thepracticability of these still very complicated machines.The demanding and sophisticated handling of such per-fusion systems may complicate the logistics and signifi-cantly increase the costs.

Hyperthermic Preconditioning

A number of animal studies indicate that the livercan be preconditioned by temporary exposure of theorgan or the whole body to hyperthermia.57–61,119 Theheat stress response is associated with the induction of anintracellular stress protein (Hsps). Experiments coveringmany organs and species showed that Hsps are inducedunder a variety of conditions of stress,120,121 includingoxidative stress (ischemia) and various pharmacologicreagents. They belong to a class of proteins called chap-erones that are involved in protein folding122 duringsynthesis and represent cellular mechanisms of protectionfrom protein degradation.

In particular, Hsp70123,124 and heme oxygenase 1 (orHsp32)78,125 contribute to the protective mechanism ofhyperthermic preconditioning, based on the finding thatoverexpression of these 2 molecules increased the resis-tance of the liver and other organs to ischemic injury.Although it is impractical to perform whole-body hy-perthermia in humans, these studies identified severalprotective pathways, which might be induced by morepractical means.


Pharmacologic StrategiesA large number of pharmacologic agents were

shown to confer protection against ischemic injury in theliver. They either blocked the injurious pathways di-rectly or they subjected the liver to preconditioning (i.e.,they induced a low level of stress to the liver cells thatinitiated cellular defense mechanisms against a subse-quent stronger insult). A nonexhaustive list of all theseagents is presented in Table 1. A number of these agentsmentioned later have been identified during studies onischemic preconditioning.

Antioxidants

There is growing evidence that the resident mac-rophages of the liver (Kupffer cells) can cause liver dam-age in a number of disease processes including cold35,36

and warm17,18,126 ischemic injury. Ischemia activatesKupffer cells, which are the main source of vascular reactiveoxygen species during the reperfusion period.127,128 Previ-ous studies had shown that newly recruited monocytesand leukocytes are partially responsible for the ischemicinjury.18,20,129 Based on their ability to kill and digestcells, macrophages also play an important role in remov-ing apoptotic bodies10,33 and in the synthesis of reactiveoxygen species17 (e.g., superoxide and hydrogen perox-ides). In hepatocytes, pro-inflammatory cytokines caninduce the formation of reactive oxygen species, for ex-ample, TNF-�, IL-1, or interferon-�.130 Moreover, isch-emic cell damage can lead to an intracellular oxidantstress during reoxygenation.131 Mitochondria are recog-nized as the major intracellular source of reactive oxygenspecies, which are generated as a product of cellularrespiration.132

The most damaging form of reactive oxygen speciesgenerated in mitochondria is the hydroxyl radical (OH�).One of the major and most sensitive targets of OH� is themitochondrial DNA.133–135 OH� attacks deoxyribose andcauses the release of nucleic acids of mitochondrial DNA,resulting in strand breaks. OH� also directly can attackbases, leading to modifications with a loss of DNAintegrity and hence lead to impaired transcription. Al-though the role of reactive oxygen species in a number ofliver diseases is generally accepted, the detailed mecha-nisms of reactive oxygen involvement are under debate.The most convincing hypothesis of reactive oxygen–induced cell injury is the destruction of cellular mem-branes through peroxidation of lipids.136 The parallelincrease of glutathione, myeloperoxidase, and products oflipid peroxidation are strongly in favor of this degener-ative process. In addition, all mitochondrial constituents,proteins, lipids, and mitochondrial DNA137 are potential

targets for reactive oxygen species–mediated damage.Through such damage, a gradual impairment of defensesin mitochondria will enhance the effect of further oxida-tive stress. In the liver, the involvement of reactiveoxygen has been suggested in apoptotic cell death ofhepatocytes and endothelial cells.138,139 One possible ex-planation is that oxidant stress can induce the mitochon-drial membrane permeability transition, a central eventpreceding cell death.140 Another potential target couldbe the caspases, a family of cysteine proteases that isimportant for the initiation and progression of apopto-sis.141 Caspases can be activated by low concentrations ofhydrogen peroxide whereas higher levels inhibit enzy-matic activity, presumably owing to oxidation of criticalsulfhydryl groups. Thus, reactive oxygen species mayinduce or inhibit apoptosis depending on the severity ofoxidative stress. The OH� concentration-dependent acti-vation or inhibition of caspases may provide an explana-tion why apoptosis and/or necrosis may both be the resultof oxidative stress.

Because of the central role of oxidative stress in thesetting of ischemia reperfusion, a large number of studies(Table 1) attempted to identify methods to either pre-vent or neutralize oxidative stress. It furthermore hasbeen shown that strategies aimed at overexpressing an-tioxidant proteins (e.g., superoxide dismutase142–144)may confer protection against extended ischemic injury.However, none of these strategies have found the wayinto routine clinical practice, with the exception of someantioxidant ingredients that were introduced into pres-ervation solutions.

Adenosine Agonists and Nitric OxideDonors

In various animal models of ischemic precondi-tioning in the heart, adenosine accumulates in the myo-cardia during ischemia and reperfusion, and confersstrong protection against ischemic injuries.145,146 Inves-tigating the protective mechanisms of ischemic precon-ditioning in the rat liver, Peralta et al.62,91,147,148 iden-tified a similar protective pathway after prolongedperiods of warm ischemia and reperfusion. In a series ofexperiments, they blocked adenosine receptors with spe-cific antagonists or metabolized endogenous adenosinewith adenosine deaminase. In both approaches, the pro-tective effects of ischemic preconditioning were abol-ished.91 Peralta et al.91 further showed that the mecha-nisms by which adenosines confer protection involves theinduction of the enzyme nitric oxide synthase in theischemic liver. This leads to increased levels of nitricoxide, which, at moderate concentrations, prevents dam-age of hepatocytes and endothelial cells.149


Table 1. Pharmacological Agents Reported to Confer Protection Against Ischemic Reperfusion Injury in the Liver

Drug Type of injury Effector system Species Reference

Oxidative stressAlbumin Cold ischemia Antioxidant Rat 216

AllopurinolWarm and cold

ischemia Oxidative stress, scavenging Rat 20,217

Atrial natriuretic peptideCold and warm

ischemia CGMP receptor–mediated oxidant stress Rat 218–220Bucillamine Cold ischemia Antioxidant Rat 221Cyclosporin, ibuprofen combined

treatment Warm ischemia Lipid peroxidation, oxidative stress? Rat 222Ebselen Warm ischemia Lipid peroxidation, oxidative stress Rat 223FK506 Warm ischemia Free radicals, suppressor of cytokine response Rat 224�-glutamylcysteine ethyl ester Warm ischemia Oxidative stress Rat 225Glycyrrhizin Warm ischemia Oxidative stress Rat 226Green tea extract Warm ischemia Free radical scavenger Rat 227Melatonin Warm ischemia Oxidative stress Rat 228Nicaravan Warm ischemia Oxidative stress Dog 229S-nitroso-�(1)-protease inhibitor

(S-nitric oxide-�(1)-PI) Warm ischemia Blood flow, heme oxygenase induction Rat 230PGI2, superoxide dismutase,

catalase, combination Warm ischemia Oxidative stress Rat 231Picroliv Warm ischemia Antioxidant Rat 232Propyl gallate Warm ischemia Antioxidant Rat 233�-tocopherol Warm ischemia Lipid peroxidation? Rat 234,235Trimetazidine Warm ischemia Scavenger of oxygen radicals Rat 236Trolox Warm ischemia Peroxyl radical scavenger Rat 237

Energy depletion mitochondriaNiacinamide Warm ischemia Adenosine triphosphate Rat 238Ozone Warm ischemia Adenosine triphosphate 239S-15176, a potent trimetazidine

derivative Warm ischemia Mitochondrial permeability transition Rat 240

Tauroursodeoxy-cholateIn vivo pig liver

transplantation Membrane stabilization Pig 241Warm ischemia Antiapoptotic? Rat 242

Microcirculation2-aminoethyl-isothiourea Warm ischemia Inducible nitric oxide synthase Rat 243

Furosemide and bumetanide Warm ischemiaNa�-K�-2Cl-cotransporter antagonist (loop

diuretics) Rat 244Misoprostol Warm ischemia Microvascular system Rat 245OP-41483 (prostacyclin) analog Warm ischemia Microcirculation Rat 182

Sodium ozagrelTransplantation,

cold ischemia Thromboxane A2 inhibitor Pig 246OKY-046 Warm ischemia Microcirculation Rat 247,248

TAK-044 Warm ischemiaEndothelin receptor antagonist,

microcirculation Dog 249TCV-116 Warm ischemia Angiotensin type II antagonist Rat 250Verapamil flunarizine Warm ischemia Ca-channel blocker Rat 251

Protease inhibitor/antiapoptoticCbz-Leu-Leu-Tyr-CHN2 Warm ischemia Calpain inhibitor Rat 32

Gabexate mesilate Warm ischemiaProtease inhibitor release of reactive oxygen

speciesRat,

human 252,253ONO-5046 Warm ischemia Granulocyte elastase Rat 254Secretory leukocyte protease

inhibitor Warm ischemia Leukocyte protease Mouse 255Urinary trypsin inhibitor Warm ischemia Myeloperoxidase, neutrophil proteases Rat 256Z-Asp-cmk (Z-Asp-2,6-

dichlorobenzoyl-oxymethylketone) Warm ischemia Caspase inhibitor Rat 84,257ZVAD-fmk Warm ischemia Caspase inhibitor Rat 165

Inflammatory and leukocyte adhesionCox-2 inhibitor Warm ischemia Anti-inflammatory Dog 258Cyclosporine/FK506 Warm ischemia Inhibition of neutrophil infiltration Rat 259Cyclosporine Warm ischemia Adenine nucleotides, mitochondrial function Rat 260

Desferriexochelin 772SM

Cold ischemiareperfusionwith blood P-selectin antagonist Rat 261

FTY20 Warm ischemia Inhibition of neutrophil infiltration Rat 262,263IL-6 Warm ischemia Anti-inflammatory Mouse 264IL-10 Cold ischemia Cytokine attenuation Mouse 48Lipopolysaccharide Warm ischemia Inflammatory preconditioning? Rat 265Prostaglandin F1 Warm ischemia Leukocyte adhesion Rat 266Ranitidine Warm ischemia H(2) receptor antagonist, neutrophil activation Rat 267Recombinant hepatocyte growth

factor Warm ischemia Inhibits neutrophil infiltration Rat 268Soluble P-selectin glycoprotein

ligand-1Warm and cold

ischemia P-selectin blocker Rat 269


This protective pathway offers several possibilities forpharmacologic interventions, including adenosine-recep-tor (A2) agonists and nitric oxide precursors (nitric oxidedonors). Several studies in animal models indicated thatsynthetic adenosine receptor agonists (e.g., CGS-21680)may confer protection to the liver against cold150 andwarm151 ischemic insults. Alternatively, administrationof nitric oxide donors such as L-arginine,149 NONOate,91

FK409,152 and others induced protection against warmischemic hepatic insults in rat models.

Pentoxifylline

Pentoxifylline is a methylxanthine theobrominederivative that has been used for many years in thetreatment of peripheral vascular disease. Pentoxifyllinehas several additional properties, which makes it anappealing candidate drug against reperfusion injury. Agenerally accepted mechanism of action is the inhibitionof intracellular elevation of cyclic adenosine monophos-phate phosphodiesterase levels, leading to increasedintracellular levels of cyclic adenosine monophos-phate.153,154

One of the interesting and important observations isthat pentoxifylline reduces TNF-� synthesis and reducessecretion of TNF-� in several organs.155–157 We haveshown that blockage of TNF-� release from Kupffer cellsby pentoxifylline prevents up-regulation of TNF-� ex-pression in a model of warm ischemic injury in themouse liver.38 In this model, inhibition of TNF-� re-sulted in a significant decrease of liver injury and markersof apoptosis and improved animal survival.38 Protectiveeffects of pentoxifylline against reperfusion injury byinhibition of Kupffer cell activation also has been re-ported in models of cold ischemia.158–160

Other mechanisms of action of pentoxifylline includeincreased red blood cell flexibility, reduction of bloodviscosity,161 and decreased potential of platelet aggrega-tion.162 These basic actions of pentoxifylline may resultin therapeutic benefits owing to improved microcircula-tion and tissue oxygenation. However, so far no clinicalstudies have been presented that would show a beneficialeffect of pentoxifylline on ischemic liver injury.

Protease Inhibitors and AntiapoptoticMolecules

Increasing evidence points to apoptosis as a crit-ical mechanism of hepatic reperfusion injury.22,33,163

Caspases belong to the family of cysteine proteinases.Specific isoforms are involved in the initiation and exe-cution phases of apoptosis. Among these isoforms,caspase 8 is activated during the early phase and caspase3 is activated during the late phase of apoptosis. Sup-

pression of their activation, or inhibition of their activity,reduces or completely abolishes apoptosis in cell culturemodels. In a rat model of warm ischemia, Cursio et al.showed maximal caspase activation 3 hours after reper-fusion, which preceded morphologic indicators of apo-ptosis.164 Pretreatment of the animals with the caspaseinhibitor Z-Asp-cmk 2 minutes before ischemia effi-ciently protected rats from lethal liver injury that nor-mally occurred 24–48 hours after surgery.84,164 How-ever, serum transaminase levels remained relatively highdespite total inhibition of caspase activities and strongsuppression of DNA fragmentation. These contradictoryfindings suggest that caspase inhibitors may completelyblock apoptosis but have little effect on necrosis. Othergroups have confirmed the protective effect of caspaseinhibition during hepatic ischemia and reperfusion insimilar or other models of cold11 and warm isch-emia.11,165 In contrast, no protection was observed in thesteatotic liver101 where the predominant form of celldeath is necrosis.

The danger of a prolonged treatment with caspaseinhibitors might include the prevention of apoptosis intissue that is not affected by the ischemic insult. Thephysiologic turnover of cells including the removal ofdefective cells or possibly even of potential cancer cellsmight be severely disturbed. This might increase thecarcinogenicity of such therapies. However, the conse-quences of antiapoptotic treatment in the setting ofischemic insults may not be significant beyond the ther-apeutic goal because this type of treatment is envisionedas a short-term therapy.

Other proteases such as calpain have been reported asmediators166 of preservation-reperfusion injury throughmodulation of apoptosis167 and necrosis.168 Calpains are agroup of nonlysosomal, cytoplasmic, calcium-dependentcysteine proteases involved in proteolysis of several cy-toskeletal and membrane proteins.169 The protective ef-fects of calpain inhibition has been reported in cold170,171

and warm32 ischemic injury. Inhibition of calpain re-sulted in decreased tissue injury in both endothelialcells12,171 and hepatocytes,170 ultimately resulting in in-creased animal survival after liver transplantation or pro-longed ischemia.

Prostaglandins

Prostaglandins are released mainly by activatedKupffer cells during the reperfusion phase.172 Animalstudies proved that prostaglandins are effective in thetreatment of ischemic liver injury owing to their abilityto increase liver perfusion, inhibit platelet aggregation,and also direct cytoprotection in a model of isolatedperfused cat liver.173 The protective action of prostaglan-


din I2 and prostaglandin E1 may be related to theirability to reduce both the release of proteases and thegeneration of oxygen free radicals from activated leuko-cytes.174,175 In addition, because of the synergistic role ofplatelets and leukocytes and the interaction of these cellswith the SECs during the reperfusion phase,30 it isconceivable that effects of prostaglandin I2 and prosta-glandin E1 on leukocyte adherence may account for theirfavorable action.176

In clinical liver transplantation, preflush of the graftwith albumin and prostaglandins before reperfusion im-proves early graft function.177 Greig et al.178 found that,after reperfusion and progression to primary dysfunction,liver function could be restored by treatment with aprostaglandin E1 analog. However, 2 randomized studiesfailed to prove a beneficial effect of a prophylactic treat-ment with a prostaglandin E1 analog; the incidence ofprimary liver failure after transplantation was not signif-icantly different in the prostaglandin E1 analog groupcompared with the placebo group, although a benefit forrenal function was observed.179,180

The use of pharmacologic doses of natural prostaglan-dins in clinical settings is limited because of drug-relatedside effects.181 Synthetic prostaglandin analogs (e.g., mi-soprostol181 or OP-41483181,182) were associated withmilder side effects and a longer half-life. Several of theseanalogs improved animal survival and prevented paren-chymal injury after prolonged periods of warm hepaticinflow occlusion.181–183 A study establishing a benefitafter transplantation and a reduction of side effects ofthese new drugs has not been presented yet.

Inhibitors of Matrix MetalloproteinaseInhibitors

Matrix metalloproteinases (MMPs) belong to thefamily of zinc-dependent metalloproteinases that are in-volved in the degradation of extracellular matrix compo-nents. Arthur et al.184 showed that several cell typesincluding Kupffer cells and probably SECs have thecapacity to secrete MMPs. A comparative study reporteda role for MMPs in cold preservation injury of the liverin humans and rats.24 The structural changes induced bycold preservation (i.e., rounding and detachment ofSECs) most probably involve alterations in the cellularcytoskeleton and in the connections between cell andmatrix.24 Changes in the extracellular MMP activities inthe early phases of reperfusion are associated with mor-phologic changes. MMPs therefore represent an appeal-ing target for pharmacologic interventions using MMPinhibitors. Interestingly, protection of the preserved liverby preservation solution can be explained partially by the

MMP-inhibiting activity of some of the ingredients (e.g.,lactobionic acid in the UW solution).24,185

Consistently, in a rat model of prolonged warm isch-emia and reperfusion, Cursio et al.186 showed that MMPsand their natural inhibitor (i.e., tissue inhibitors ofMMPs) genes are induced in a specific time-dependentmanner after ischemia and reperfusion, suggesting thatMMPs and tissue inhibitors of MMPs could play bothdeleterious and beneficial roles. Pretreatment of the an-imals with the phosphinic MMP inhibitor RXPO3 sig-nificantly reduced markers of parenchymal injury andapoptosis.

Unfortunately, clinical trials with several MMP inhib-itors for the treatment of cancer or chronic inflammatorydiseases have led to disappointment, including unfavor-able side effects, suggesting that chronic pharmacologicapplication of such molecules is not advisable at thispoint.187,188 However, because protective strategiesagainst ischemia reperfusion injuries will not requirechronic application of MMP inhibitors, such drugs maybe valuable in the treatment of acute processes such ascold or warm hepatic ischemia.

Cooling of the Organ and PreservationSolutions

Reduction of metabolic activities by cooling ofthe organ to 1°C to 4°C was among the first strategiesdesigned to protect against ischemic injury.4 This strat-egy may safely preserve the liver for transplantation forup to 8 hours, whereas livers kept at room temperaturetolerate only about 1 hour of ischemia before reperfusion.Cooling requires the application of a perfusion/preserva-tion solution. Thus, efforts were directed at designing aneffective solution that would extend the period for safepreservation. The breakthrough in this field came in themid-1980s as a result of the lifelong work of Belzer andSouthard.4 They empirically designed a solution based onthe known and speculated negative effects of hypother-mia. These negative effects include (1) cell swellingcaused by inhibition of the membrane pump Na�/K�

adenosine triphosphatase regulating cell volume189; (2)intracellular acidosis caused by anaerobic metabolismand lactate accumulation190; (3) disturbances in the ho-meostasis of cytosolic Ca��191; and (4) free radical gen-eration.192,193 Belzer and Southard4 included severalagents in their UW solution, each of them presumed tocounteract the potential negative effects of hypothermia.Although this solution represents one of the most sig-nificant progresses in the field of liver transplantation byextending safe preservation beyond 24 hours (comparedwith 8 hours with Euro Collins solution),8,194 the mech-anisms involved in protection remain largely unknown.


The UW solution was shown to protect mainly againstSEC injury.13,195 Therefore, it was speculated that thesolution exerted its protective effect by inhibition ofMMP activity owing to one of the ingredients (lacto-bionic acid), a potent MMP inhibitor.185 Recently,Bretschneider’s196 solution, also known as histidine/tryp-tophan/ketoglutarate solution, has been shown to beequally effective as the UW solution at the usual periodsof cold preservation used in human transplantation.197,198

This is a surprising outcome because the compositions ofthese 2 solutions are very different, and, seemingly, theonly property shared by these solutions is bufferingcapacity and MMP inhibition. However, buffering ca-pacity alone cannot explain their effectiveness becausesolutions with excellent buffering capacity, such asKrebs-Henseleit solution and Euro-Collins solution, arepoor preservation solutions. These results underline thelack of understanding of protective mechanisms of pres-ervation solutions. Strategies aimed at improvement ofcurrently used preservation solutions by adjunction ofbetter pharmacologic agents should be a goal of futureinvestigations but are not the main focus of this article.

Gene Therapy

Recent advances in genome manipulation providenew tools to reduce or suppress liver injury by genetherapy. Genome manipulation can be achieved by: (1)manipulation on the germ line (e.g., oocyte injections),(2) stem cell transformation and reintroduction into em-bryos, and (3) targeting specific cells or organs withvectors or viruses (e.g., adenovirus) carrying a gene ofinterest. The first 2 approaches may include germ-linealterations and are neither feasible nor accepted by soci-ety. The third approach, representing somatic therapy,would lend itself to the treatment of individual patientswith either acquired or congenital diseases. In the pastdecade, efforts toward construction of cell or organ-specific virus with a high infectivity rate,199 transgeneexpression,200 and replication deficiency have resulted ina battery of tools with which gene therapy may beenvisioned.201 Modified recombinant adenovirus now al-low specific targeting to the liver without affecting otherorgans.

With respect to liver transplantation, pretreatment ofdonors with such vectors might help to suppress thereperfusion injuries of the liver. However, liver trans-plantations are in most cases an emergency procedure(cadaveric livers), leaving very little time to pretreat thedonor with genetic approaches. In the situation of living-related donors, the elective nature of the procedure mayallow a pretreatment protocol with such vectors. Again,the ethical aspect has to be taken into consideration: a

healthy subject (the donor) has to be treated beforesurgery.

Some of the target genes that have been introducedsuccessfully in animal experiments with the goal of sup-pressing ischemia reperfusion injury are discussed later.

Antiapoptotic Strategies: Bcl-2 and Bag-1

Members of the family of proteins related to theoncogene Bcl-2 are potent regulators of the programmedcell death and either can prevent (e.g., Bcl-2 or Bcl-xL) orpromote (e.g., Bid, Bad, or Bax) the apoptotic path-ways.202–207 The antiapoptotic mitochondrial proteinBcl-2 is the best characterized member of the Bcl-2family and has been studied in the setting of hepaticischemia.85,86 In mouse models of prolonged hepaticischemia, overexpression of this protein (either by usingtransgenic mice85 or by adenoviral transfection86) wasassociated with strong protection against reperfusion in-jury. Biochemical and morphologic evaluation of celldeath revealed that the number of apoptotic hepatocyteswas reduced significantly. In addition, increased expres-sion of Bcl-2 prevented mortality of animals after pro-longed ischemic insults. Although in the liver no in-volvement of this protein in the mechanisms of ischemicpreconditioning was found,76 Bcl-2 appears to play a rolein protecting the preconditioned heart against myocar-dial infarction.208

The Hsp70/Hsc70 chaperone regulator Bag-1209 alsohas been shown to be a powerful antiapoptotic proteinthat appears to interact with members of the Bcl-2oncogene family. Bag-1 is a Bcl-2 binding protein re-sulting in a prolonged and stabilized antiapoptotic ac-tivity.210 In addition, Bag-1 appears to exert an indirectsilencing effect on TNF receptor R1 and hence sup-presses the death receptor signal. Sawitzki et al.211 useda model of cold ischemia and orthotopic liver transplan-tation in the rat to test the effect of adenoviral-mediatedtransfer of the Bag-1 gene. In contrast to an unrelatedgene, �-galactosidase, the adenoviral Bag-1 constructhad a beneficial effect on the histopathology of the grafts,particularly on the extent of necrosis. Indicators of in-flammation, TNF-�, CD25, IL-2, and interferon �, werereduced on the messenger RNA level. Finally, survival ofthe recipient animals was increased from 50% to 100%.These results are highly encouraging to develop such apowerful technique.

Antioxidant Therapy

Ischemia and reperfusion injuries are character-ized by a burst of oxygen radicals leading to increased


apoptosis of hepatocytes (for a detailed description ofoxidative stress–induced injury see Pharmacologic Strat-egies). To suppress the burst of reactive oxygen species orits effect on hepatocellular activation of nuclear factorkappa B, several groups have tried to introduce inhibi-tory proteins in the stress response pathway. The firstprotein, mitochondrial superoxide dismutase, was trans-fected in mice by adenoviral gene therapy.142 Using amodel of partial hepatic ischemia and reperfusion injuriesa beneficial effect of the treatment was shown. Subse-quent studies by others showed that the introduction ofgenes coding for cytosolic as well as mitochondrial su-peroxide dismutase successfully reduced warm ischemiareperfusion injury.144 Surprisingly, intravenous applica-tion of an adenovirus coding for extracellular superoxidedismutase only was effective at a high rate of infection.

Another protein, heme oxygenase 1, also known asHsp32, appears to be induced by various conditions suchas hypoxia212, 213 and hyperthermia.214 In several exper-imental approaches it was shown that heme oxygenase 1exhibits a cytoprotective effect after hyperoxia or afterischemia and reperfusion. In an effort to develop strate-gies to expand the donor pool, Coito et al.125,215 used thegene therapy approach in Zucker rats. In contrast tocontrol vectors, pretreatment of donor rats with adeno-viral vectors carrying the heme oxygenase 1 gene wereable to significantly improve several parameters afterwarm ischemia and orthotopic liver transplantation. Sur-vival of the recipient was increased from 40% to 80%,whereas necrosis, edema, and macrophage infiltrationwere decreased markedly by the treatment. Although themechanism of protection remains unclear, proteins withknown antiapoptotic activities (e.g., Bag-1 and Bcl-2)were increased whereas inducible nitric oxide synthetasewas reduced.125 The latter has been associated with isch-emic injury because increases in enzymatic activity havebeen found in liver injuries induced by oxidative stress.

Although gene therapy approaches have been usedpredominantly in experimental studies they appear toprovide an elegant alternative strategy to induce protec-tive mechanisms. If similar protocols as used in experi-mental settings would be applied to the clinical situationthe donor would have to be treated with the virus 24hours before transplantation. This temporal limitationthus restricts this potential therapy to a subgroup ofdonors (i.e., living-related donors). However, treatmentof a healthy donor with adenoviral vectors with its po-tential negative side effects is currently ethically unac-ceptable. Efforts to reduce the time between gene ther-apy and transplantation might open new venues forpreventative gene therapy.

Outlook on Possible Practical Applications

In our view, among the various pharmacologicapproaches mentioned earlier, only a few drugs are cur-rently at the point of clinical application. Pentoxifyllinecould be one potential agent in the near future. Thisdrug currently is used safely in clinical setting of variouspathologies such as inflammatory bowel disease. Otherdrugs such as prostaglandins and MMP inhibitors haveno proven beneficial effects in clinical trials despite en-couraging results in animal models. Finally, caspase in-hibitors present a potential danger of cancerogenesis byinhibition of apoptosis. Even though chances are slimthat these inhibitors promote the development of can-cerous growth in other tissues, their eventual use in theclinic should be monitored carefully. In the same setting,the gene therapy strategies are still far beyond introduc-tion into clinical practice.

Currently, only surgical strategies could be consideredas protective strategies against ischemic reperfusion in-jury routinely used in clinical practice. All other modal-ities need to be better evaluated in phase I studies beforeroutine use.

In conclusion, the complex mechanisms of hepaticinjuries encountered in various clinical situations havespawned a battery of different approaches to developprotective strategies. The most promising strategies todate are intermittent clamping and ischemic precondi-tioning, which are used during surgery. In addition,pharmacologic strategies with the goal to prevent inju-ries have led to the identification of dozens of promisingdrugs, most of which have not reached a clinical appli-cation. Finally, targeting the liver by gene therapy is apromising new tool that requires ethical discussion be-fore reaching the clinical level.

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Received December 23, 2002. Accepted May 22, 2003.Address requests for reprints to: Pierre-Alain Clavien, M.D., Ph.D.,

Department of Visceral Surgery and Transplantation, University Hospi-tal of Zurich, Ramistrasse 100, 8091 Zurich, Switzerland. e-mail:[email protected]; fax: (41) 1-255-44-49.

Supported by a grant from the National Institutes of Health(DK54048) and from the Swiss National Science Foundation(SNF3200-061411) (to P.-A.C.).

N.S. and H.R. contributed equally to this work.


Protective strategies against ischemic injury of the liver

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