a therapeutic cocktail with a manifold ameliorative effect on hepatic ischemia_reperfusion injury.

Fundamental Efforts toward the Development of a Therapeutic Cocktail with a Manifold Ameliorative Effect on Hepatic Ischemia/Reperfusion InjuryGENEVIEVE SCHINDLER,*MARIUS KINCIUS,*RUI LIANG,*JU¨RGEN BACKHAUS,$MARKUS ZORN,%CHRISTA FLECHTENMACHER,§MARTHA-MARIA GEBHARD,*MARKUS W.BU¨CHLER,*AND PETER SCHEMMER**Departments of General Surgery;$Pharmacy;§Pathology;%Central LaboratoryRuprecht-Karls-University,Heidelberg,GermanyABSTRACTObjective:Ischemia/reperfusion injury is mediated by various mechanisms.The present study was designed to evaluate the effect of a multiple pharmacological approach toward ischemia/reperfusion injury reduction.Methods:The left liver lobe of Sprague-Dawley rats underwent normothermic ischemia for 90minutes after a cocktail (glycine,taurine,alanine,arginine,and prednisolon)intravenous administration.Controls received normal saline.Liver injury (transaminases,histology)and cellular activation [Kupffer cell phagocytosis,production of tumor necrosis factor-alpha (TNF a ),and prostaglandin E-2(PGE 2)],as well as microcirculation and leukocyte-endothelial interaction (in vivo microscopy),were assessed.Results:Whereas in controls a substantial increase of aspartate aminotransferase,alanine aminotransferase,and lactate dehydrogenase to 2,3419477,1,4429262,and 1,973973U/L,respectively,was measured eight hours after reperfusion,the cocktail significantly reduced the increase of enzymes by 33Á80%(P B 0.05).Further,necrosis,index of liver damage,and index of leukocyte infiltration significantly decreased after the cocktail (P B 0.05).Moreover,the cocktail improved acinar and sinusoidal perfusion,while the sinusoidal diameters,leukocyte-endothelial interaction,Kupffer cell phagocytic activity,and TNF a /PGE 2serum levels were significantly reduced,the latter by 86%and to 1.64-fold,respectively.Conclusions:This study depicts that multifaceted pharmacological tackling of ischemia/reperfusion injury is feasible and protects rat liver tissue from warm ischemia/reperfusion injury.Microcirculation (2009)16,593Á602.doi:10.1080/10739680903110779K EY WORDS :ischemia,reperfusion injury,liver,microcirculation,cocktail,glycine,taurine,arginine,alanine,prednisolonINTRODUCTIONIschemia/reperfusion injury (IRI)occurs during a multitude of surgical procedures,including liver resection and liver transplantation,which are,in turn,accompanied by various degrees of hepatic damage,leading to organ dysfunction or failure.The substantial contribution IRI renders to the morbidity and mortality after liver surgery still remains persistent,although significant efforts toward the understanding of the underlying mechanisms of IRI have been made over recent decades.The complexpathophysiology of IRI has resulted in the sprawling of a battery of different approaches toward achiev-ing liver protection.Various reviews of IRI conclude that due to the diverse mechanisms that contribute,to a degree,to the overall pathophysiology of IRI,it is difficult to attain effective protection when target-ing individual mediators or mechanisms [38,13].Numerous promising attempts to enhance liver via-bility following IRI include reducing hepatic micro-circulation disturbance [16,37,14,32],ameliorating the various Kupffer cell Áderived proinflammatory mediators,such as tumor necrosis factor-alpha (TNF a )and prostaglandin E-2(PGE 2)and free radicals [36,21,31,33,5,34,15],as well as down-regulating the recruitment of neutrophils and lymphocytes [21].Most single drugs with significant protective effects against IRI have not attained the level of clinical routine yet [38,35];this may probably be the result of the potential toxicityAddress correspondence to Peter Schemmer,Department of General Surgery,Ruprecht-Karls-University,Im Neuenheimer Feld 110,69120Heidelberg,Germany.E-mail:Peter.Schemmer@med.uni-heidelberg.deReceived 23October 2008;accepted 11June 2009.Microcirculation,16:593Á602,2009Copyright #2009Informa UK Ltd.ISSN:1073-9688print /1549-8719online DOI:10.1080/10739680903110779of the hepatoprotective compounds themselves or a course of the special mode of administration required for their successful application.Most recently,glycine,a nontoxic amino acid,was proven to reliably protect livers from experimental IRI and to improve graft function and survival after transplantation via prevention of Kupffer cell activation,most likely by inactivating glycine-gated chloride channels[37,31,49].Taurine,a sulfur-containing b-amino acid,acts similarly to glycine in Kupffer cells.Taurine causes hyperpolarization, which blocks the lipopolysaccharide(LPS)-induced increase of internal calcium ion concentration [Ca2']i and TNF a production by mechanisms involving chloride influx into Kupffer cells,and it is also a potential radical scavenger[15,11,10]. L-arginine,a precursor required for nitric oxide (NO)synthesis,is reported to induce protection against warm ischemic hepatic insults in rats, probably by improving microcirculation through the induction of vasodilation at the sinusoidal level and at presinusoidal sites[13,16,44,29].Further, NO protects mitochondria and induces heat shock proteins,which are known to confer cytoprotection against ischemia through reactive oxygen species (ROS)depletion[13,29,46].NO also inhibits apoptosis by mechanisms that include the direct inhibition of caspases by nitrosylation of the active site[29].Santiago-Delpin et al.first described the protective effects of steroid application prior to IRI in rabbits and in humans[30,8,28];based upon their findings, steroid treatment is used in clinical liver surgery routinely[47].Recently,it was reported that postischemic apoptotic activity and inflammatory response can effectively be decreased by means of steroids[9].Despite controversial results[9,43], high glycogen content within donor liver seems to be beneficial to the outcome of transplantation in rats and humans[2].The beneficial effects of alanine administration to hepatic metabolism and energy status have been investigated in both models of cold ischemia and transplantation[2,1].It can thus be implied that the application of L-alanine may attenuate IRI by stabilizing the hepatic energy household.This study was,therefore,designed to tackle the complex pathophysiology of IRI by administering a drug cocktail containing glycine,taurine,L-alanine, L-arginine,and prednisolon,all promising sub-stances that have proven to ameliorate the major injury mechanisms involved in IRI.The concentra-tions of the various components of the cocktail were chosen on the bases of concentrations that have been proven to be most advantageous in the literature,as stated above.Our aim was to investigate the in vivo effect of this cocktail on microcirculation,leukocyte-endothelial interaction,activation of Kupffer cells, as well as proinflammatory mediator release. MATERIALS AND METHODSAnimalsAll experiments were conducted in accord with the German Legislation on Laboratory Animal Experi-ments.Female Sprague-Dawley rats(200Á250g) were housed in a temperature-and humidity-con-trolled room under a constant12-hour light-dark cycle.Before experiments,all animals were allowed free access to standard laboratory chow(ssniff R/M-H;ssniff Spezialdia¨ten,Soest,Germany)and tap water.All animals received humane care according to institutional guidelines.Liver Ischemia Reperfusion Model/Surgical ProcedureRats were anesthetized with intraperitoneal(i.p.) Narcoren†(20mg/kg body weight;pentobarbital-natrium16.0g,benzylalkohol3.0g in100mL of Narcoren;Merial GmbH,Hallbergmoos,Germany), and intramuscular(i.m.)Ketanest†(100mg/kg body weight;Esketaminhydrochlorid;Parke-Davis GmbH,Berlin,Germany),after which polypropy-lene catheters(Braun,Melsungen,Germany)were placed in the left carotid artery for hemodynamic surveillance and in the right jugular vein for infusion administration.Body temperature was kept between 36.5and37.58C by means of a heating pad. Following laparotomy,a Yasargil clamp(Aesculap, Tu¨bingen,Germany)was used to interrupt the arterial and portal blood supply to the left lateral lobe.The remaining lobes retained an intact portal and arterial blood supply as well as venous outflow,preventing the possible leakage of bacteria or bacterial products into the circulation.After 90minutes of warm ischemia,the clamp was removed to initiate reperfusion.Preliminary titration experiments were carried out to prove whether the concentrations of the various cocktail components stated in the literature workedCocktail to abate ischemic hepatic injury 594G Schindler et al.in our hands as well.Based on these findings, we then established the cocktail by adding,to a 300.0-mM glycine base solution,11.4mM of taur-ine,14.6mM of alanine,114.8mM of arginine,and 0.7mM of prednisolon(Department of Pharmacy, University Hospital of Heidelberg,Germany).To elucidate the beneficial effects of the therapy cock-tail,two animal groups(control vs.cocktail group; n08Á10each)were assessed.The cocktail group received1.5mL of the prepared cocktail,consisting of300.0mM of glycine,11.4mM of taurine, 14.6mM of alanine,114.8mM of arginine,and 0.7mM of prednisolon(Department of Pharmacy, University Hospital of Heidelberg,Germany) 15minutes prior to ischemia,whereas controls received normal Ringer’s solution,according to the same schedule.To further test the hypothesis that a cocktail potentially reduces IRI to a greater extent, compared to the single substances,the left liver lobe of rats was subjected to normothermic ischemia for90minutes and preconditioned with either i.v. glycine or taurine or glycine'taurine in concentra-tions and doses,as in the therapy cocktail.Eight hours after reperfusion,an intravital microscopic examination was performed.Blood samples were collected,centrifuged,and stored atÁ208C until the determination of plasma aspartate aminotransferase (AST),alanine aminotransferase(ALT),lactate dehydrogenase(LDH),TNF a,and PGE2.Ischemic liver tissue obtained at animal sacrifice was fixed in 4%formalin for subsequent histological study.In Vivo Fluorescence MicroscopyHepatic microcirculation was observed in vivo,as described elsewhere[45,4].Briefly,the surface of the left liver lobe was epi-illuminated with the 100-W mercury lamp of a modified inverted Leitz-Orthoplan microscope(Leitz,Germany),using two filter sets:a450-to490-nm excitation and a515-to560-nm emission band-pass filter.For fluorescent staining,fluorescein isothiocyanate(FITC)-labeled red blood cells(RBCs)(3m mol/kg body weight; FITC Isomer1;Sigma-Aldrich,St.Louis,MO,USA Germany)and rhodamine6G(0.05mmol/kg body weight;Sigma),for staining leukocytes,were admi-nistered simultaneously.At first,the FITC filter was used to asses the number of perfused acini at10X objective,then the sinusoidal perfusion,sinusoidal diameter,as well as permanent adherent leukocytes (stickers)in10Á15randomly selected acini and 5venules were observed in each animal at40X,with the aid of the excitation filter.At last,fluorescent latex beads(3)108beads/kg body weight,dia-meter01.1m m;Polysciences Inc.,Warrington, Pennsylvania,USA)were injected intra-arterially, allowing an indirect analysis of the activation of Kupffer cells by an immediate count of the number of latex beads phagocytized by Kupffer cells per microscopic field for300seconds.Beads in hepatic arterioles,portal venules,and postsinusoidal venules were not counted.The microcirculatory images were transmitted by a video camera(CF8/1;Kappa GmbH,Gleichen, Germany)to a monitor(PVM-1440;Sony,Tokyo, Japan)and recorded per video recorder(AG-7350-E;Panasonic,Osaka,Japan)for off-line evaluation. Quantitative Off-Line Video AnalysisThe recorded images were analyzed during video playback,using special software(Capimage;Zeitl Gmbh,Heidelberg,Germany).The acinar perfusion index was calculated with the following formula: (NG'0.5NI)/NT[45].NG represents the number of well-perfused acini,NI the number of irregularly perfused acini,and NT the total number of analyzed acini.Sinusoidal perfusion was quantified in10 randomly selected microscopic areas per animal and results displayed as percent of perfused sinusoids [15,4].The sinusoidal diameter was measured directly in40different sinusoids per animal,and the mean calculated stickers were defined as leuko-cytes adhering for more than20seconds to the endothelium[23].Liver and Hepatocellular DamageAs an indicator of hepatocellular injury,the plasma AST,ALT,and LDH activities were determined by standard enzymatic methods[3].HistologyLiver specimens were fixed by perfusion with4% paraformaldehyde in Krebs-Henseleit bicarbonate buffer(118mM NaCl,25mM NaHCO3,1.2mM KH2PO4,1.2mM MgSO4,4.7mM KCl,and1.3mM CaCl2)at pH7.6and embedded in paraffin.Liver sections(4m m)were stained with hematoxylin and Cocktail to abate ischemic hepatic injury595 G Schindler et al.eosin and then evaluated with a light microscope. The histomorphological severity of IRI was graded by using a point-counting method,as described elsewhere[39].Briefly,40sections,each0.15mm2, were evaluated per slide by using an ordinal scale as follows:grade0,minimal or no evidence of injury; grade1,mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis;grade2, moderate to severe injury with extensive nuclear pyknosis,cytoplasmic hypereosinophilia and loss of intercellular borders;and grade3,severe necrosis with disintegration of hepatic cords,hemorrhage, and neutrophil infiltration.Forty sections were investigated per slide to determine the percentage of necrotic cells.To index leukocyte infiltration to the liver vasculature,a semiquantitative scale for each hepatic zone from zero to four was used:grade 1,B10leukocytes/40X field(focal infiltration); grade2,10Á20leukocytes/40X field(mild infiltra-tion);grade3,21Á50leukocytes/40X field;and grade4, 50leukocytes/40X field.Enzyme-Linked Immunosorbent Assay(ELISA) ELISA kits were used to determine the serum TNF a (eBioscience,San Diego,California,USA)and PGE2 (R&D Systems GmbH,Wiesbaden-Nordenstadt, Germany)levels.All samples were tested in dupli-cates,according to the manufacturer’s instructions, and the absorbencies of the samples were compared with standard curves.Statistical AnalysisAll values are expressed as mean9standard error of the mean.Data were first analyzed by using one-way analysis of variance(ANOVA)with the Bonferroni post-hoc test for parametric data.Differences in histological grading of injury were tested by ANOVA on ranks.A value of P B0.05was selected prior to the study as the criterion for significance of differences between groups.RESULTSMacrohemodynamicsBlood pressure(10492mmHg),hematocrit(46.49 2.3%),and body temperature(36.090.28C)were comparable in both groups during all experimental phases studied.Hepatocellular Injury after Warm IschemiaThe extent of parenchymal cell damage following 90minutes of warm ischemia was depicted by the increase of serum AST,ALT,and LDH levels in controls to2,3419477U/L,1,4429262U/L,and 1,973973U/L,respectively,at eight hours after reperfusion.Glycine or taurine or glycine'taurine decreased AST levels to652964U/L,8399124U/ L,and5599158U/L;ALT levels to397975U/L, 7449134U/L,and3889128U/L;and LDH levels to154916U/L,369951U/L,and229940U/L eight hours after reperfusion,respectively.The enzyme release in the cocktail group was decreased by33Á80%of control levels at eight hours of reperfusion time(Figure1).Reduced liver cell injury after infusion of the cock-tail is further supported by the histological analysis eight hours after reperfusion(Table1,Figure2). Histological pictures demonstrated the substantial prevention of necrosis eight hours after reperfusion, in both pericentral and midzonal areas in the cocktail group(Figure2B),compared to controls (Figure2A).Further,histological indicators of IRI examined at eight hours after reperfusion showed analogous results to the findings mentioned above; both the index of liver damage and leukocyte infiltration were significantly reduced from values of2.790.09and3.290.3,respectively,in controls to 1.190.08and 1.790.1,respectively,in the cocktail group(Table1).Effect of the Cocktail on Hepatic Microcirculation The cocktail application resulted in a better acinar perfusion index eight hours after reperfusion. Similarly,the cocktail could improve the sinusoidal perfusion rate significantly at eight hours from 77.192.0%in controls to85.592.9%in the cocktail group(Table2).Sinusoidal DiameterThe mean sinusoidal diameter was significantly larger in the cocktail group(10.990.3m m),Cocktail to abate ischemic hepatic injury 596G Schindler et al.compared to controls (8.790.6m m)(P B 0.05)(Table 2).Leukocyte-Endothelium InteractionIn vivo microscopy revealed considerable leukocyte adherence within sinusoids of all three evaluated zones (periportal,midzonal,and pericentral)in all animals (Table 2);however,cocktail administration could counteract the permanent sticking of leuko-cytes to the sinusoids by 45,35,and 52%in the periportal-,mid-and pericentral zones,respectively (Table 2).PhagocytosisAs an indirect marker of Kupffer cells activation at eight hours reperfusion time,the number of latex beads phagocytized by Kupffer cells was compared in both groups.The cocktail decreased the number of latex beads after ischemia-reperfusion from 26911/mm 2in controls to 994/mm 2,thus minimizing the Kupffer cell activation significantly (Figure 3A).Proinflammatory MediatorsThe cocktail significantly decreased the proinflam-matory mediator production after reperfusion.The PGE 2expression showed more than 1.64-fold higher mediator levels in controls,compared to the cocktail group (Figure 3B).The serum TNF a value showed analogous findings of significant reduction by about 86%of that of the control group (Figure3C).Figure 1.Effects of the cocktail on aspartate aminotrans-ferase (AST),alanine aminotransferase (ALT),and lactate dehydrogenase (LDH).Before initiating warm ischemia,animals were infused with 1.5mL of either therapy cocktail (300.0mM of glycine,11.4mM of taurine,14.6mM of alanine,114.8mM of arginine,and 0.7mM of prednisolon)or Ringer ’s solution.Immediately,three and eight hours after reperfusion,serum parameters (A,AST;B,ALT;and C,LDH)were determined,as described in Materials and Methods.Values are mean 9standard error of the mean (P B 0.05by one-way analysis of variance with Bonferroni post-hoc test;n 08Á10).*P B 0.05for comparison to corresponding controls.Table 1.HistologyParameter Control 8Cocktail 8Index of liver damage 2.790.09 1.190.08*Index of leukocyte infiltration 3.290.3 1.790.1*Area of necrosis [%]37.4910.710.891.5*Before initiating warm ischemia,animals were infused with 1.5mL of either the therapy cocktail (300mM of glycine,11.4mM of taurine,14.6mM of alanine,114.8mM of arginine,and 0.7mM of predniso-lon)or Ringer ’s solution.Eight hours after reperfusion,liver tissue was taken and processed for light microscopy by hematoxylin and eosin staining.The degree of hepatic injury and leukocyte infiltration was determined by a point-counting method as,described in Materials and Methods.Control 8,cocktail 8;eight hours after reperfusion.Values are mean 9standard error of the mean;*P B 0.05by one-way analysis of variance.Cocktail to abate ischemic hepatic injury 597G Schindler et al.DISCUSSIONThe current consensus on IRI is that it has a complex pathophysiology,with numerous contributing fac-tors.IRI contributes to the morbidity and mortality associated with many surgical procedures,including hepatic resection and transplantation,and its under-lying mechanisms tend to be interrelated.Although lots of research has been undertaken,to date,to investigate and find productive strategies to amelio-rate IRI,none among the many drugs proposed has reached the level of clinical application [38,35].This may be,in part,because of the multifaceted nature of IRI,hence requiring more than just a single substance,which sometimes reaches a toxic dose before achieving satisfying results.Kupffer cell activation,upon reperfusion after ischemia,has been identified as a key event in the initiation and perpetuation of IRI injury [20].Kupffer cells disturb the intrahepatic circulation [16,37,14,32]by releasing vasoactive mediators [36,21,33,15].Similar to adhesive and activated white blood cells,activated Kupffer cells are,perhaps,the most important source of ROS and proinflammatory mediators involved in the development of reperfusion injury [14,21,20].In addition,accumulation of leukocytes,in both hepatic sinusoids and in postsinusoidal venules following reperfusion of ischemic livers,has been reported [45,20].Adhesion molecules,such as selectins and integrins,are expressed on vascular and leukocyte membrane surfaces.The leukocyte adhesion in venules can be induced after ischemia by mediators,such as TNF a ,partly derived from activated Kupffer cells.Glycine [12]as well as taurine [36]have been reported to reducetheFigure 2.Histology eight hours after reperfusion.Con-ditions,as described in Figure 1.Liver tissue was taken eight hours after reperfusion and was processed for light microscopy by hematoxylin and eosin staining.(A )control;(B )cocktail.Necrosis was focused pericentral and midzonal depicting typical pattern of injury.Table 2.Microvascular Perfusion,Sinusoidal Diameter,and Adherence of LeukocytesParameterControl 8Cocktail 8Sinusoidal diameter (m m)8.790.610.990.3*Adherence of leukocytes in sinusoidsStickers in zone 1(periportal)(n/mm 2)53.295.129.195.1*Stickers in zone 2(midzonal)(n/mm 2)59.497.338.794.2*Stickers in zone 3(pericentral)(n/mm 2)65.496.731.292.9*Acinar perfusion index0.790.090.990.04Sinusoidal perfusion rate (%)77.192.085.592.9*Sinusoidal and acinar perfusion were evaluated at eight hours reperfusion time (control 8and cocktail 8).Values are mean 9standard error of the mean (P B 0.05by one-way analysis of variance with the Bonferroni post-hoc test;n 08Á10).*p B 0.05for comparison to corresponding controls.Cocktail to abate ischemic hepatic injury598G Schindler et al.activation of Kupffer cells by possibly acting on chloride channels and subsequently preventing the [Ca 2']i increase.Besides its hepatoprotectiveproperties,glycine is further known to exert protec-tive effects on kidney,heart,and intestine [49].The role of taurine as an antioxidant,in conjugation reactions and as a membrane stabilizer,has been described.The antioxidative effect of taurine is based on the formation of chloramines.Osmolytes,including taurine,have been shown to stabilize protein structures by the prevention of denaturation [48].Further,taurine modulates the expression of heat shock protein 70and the balance between c-Jun N-terminal kinase-2and mitogen-activated protein kinase phosphatase-1activity [19].Thus,the interaction with intracellular signal-transduction pathways may be a further explanation for the protective effect of taurine in models of IRI.There-fore,although glycine and taurine may have an identical action on Kupffer cells,as already described,their incorporation into our study cocktail seems reasonable.The decrease in latex beads phagocytosis and the reduction in cytokine release in the cocktail group,compared to controls (Figure 3),implicates that IRI attenuation may have been achieved,in part,by less activation of Kupffer cells.NO is reported to have a potentially protective effect against IRI in the liver [16,44].NO possibly reduces apoptosis by inhibiting caspase activation through nitrosylation of the active sites [29].Microcircula-tory disturbances and nonperfused sinusoids are partly responsible for reperfusion injury after hepatic ischemia [45,17],with active vasoconstric-tion being a major contributor [24].NO,a potent vasodilator,diffuses freely across membranes and acts intracellularly by the activation of guanylate cyclase.It,then,induces vasodilation in response to vasoconstrictors at the level of sinusoids and at presinusoidal sites [24,25].Further,NO protects mitochondria and induces heat shock proteins [46].L-arginine,a precursor required for endogenous NO synthesis [6],has been reported to be depleted after the onset of reperfusion through the release of arginase,which breaks down L-arginine [29].The fact that significant production of NO is unlikely to occur until six hours postreperfusion makes the pharmacological exogenous means of enhancing the NO concentration advantageous [29,40,41].The above elaborated strategies may explain why the sinusoidal diameters of the cocktail-treated group showed better values,compared to controls.The use of sinusoidal diameters as an outcome measure for the effect of NO has been described elsewhere [48].The improvedmicrocirculationFigure 3.Kupffer cell phagocytosis and proinflamma-tory mediator release.(A )Conditions,as described in Table 1.At the end of intravital microscopy,an intra-arterial injection of fluorescent latex beads was performed to study their phagocytosis by Kupffer cells.The number of latex beads Ápositive Kupffer cells was counted per mm 2.The high number of beads taken up by Kupffer cells in control animals suggests their activation.This was significantly reduced by the cocktail.(B and C )The serum levels of tumor necrosis factor-alpha and prostaglandin E-2were analyzed at eight hours reperfusion time.Values are mean 9standard error of the mean (P B 0.05by one-way analysis of variance with Bonferroni post-hoc test;n 08Á10).*P B 0.05for comparison to controls.Cocktail to abate ischemic hepatic injury 599G Schindler et al.and subsequent reduced liver damage(Tables1and 2;Figures1and2)may be an additional evidence of the favorable effects of NO.IRI triggers a number of cellular cascades,which,in turn,make use of energy stores.Fasting is known to be a condition deleterious for hepatic function and recovery after cold ischemia [1].Infusion of glucose to donors during explanta-tion is shown to improve the outcome of transplan-tation in livers with low glycogen stores in both experimental and clinical studies[26,7].However, considering complications,such as hyperglycemia and/or hyperosmolarity,routine administration of glucose to counteract IRI-induced energy depletion in hepatic surgery is controversial[43,42].Upon this viewpoint,the application of a gluconeogenesis precursor,such as L-alanine,seems reasonable[1]. L-alanine has further been reported to interact with membrane ion transporters,including chloride channels[22,18],thus playing a role in reducing IRI through cellular protection.Here,the question as to whether this mechanism of action is not similar to that of glycine,and if there is no interaction between the two substances,arises.Probably,the effects of the two amino acids are mediated in different ways, as glycine seems to be protective for short fasting periods,while alanine does not[1].The effect of L-alanine,in our study,may,however,only be explained indirectly through the generally reduced liver damage in the cocktail group,depicted by less transaminases elevation and better histological findings,compared to controls(Table1,Figures1 and2).Recently,methylprednisolone has been reported to effectively reduce the postischemic apoptotic activity and inflammatory response,probably through the reduction of NF-k B-binding activity [25].Our cocktail contains only1mg/kg body weight of the prednisolone(Solu-Decortin†;Merck Pharma GmbH,Darmstadt,Gemany),which is far less than the methylprednisolone amount;3mg/ 100g body weight reported to be effective in attenuating IRI[9].Even so,this lesser amount of prednisolone seemed to reduce inflammation,as shown by improved microcirculation,in terms of improved acinar perfusion(Table2).The under-lying mechanism may probably be a reduction in cell swelling,especially of Kupffer cells.The remarkable drop of the liver-unspecific LDH levels in cocktail vs.the control group(Figure1C)may mirror the prevention of extrahepatic tissue damage, which contributes to IRI.CONCLUSIONS AND FUTURE IMPLICATIONSThe present study demonstrates the actions of atherapy cocktail toward the reduction of IRI,deli-neated by the reduction of liver injury and inflam-mation,improvement of microcirculation,inactivation of Kupffer cells,reduction of leukocyterecruitment,and activation and the subsequent prevention of proinflammatory mediator release.Afurther advantage of this multiple pharmacologicalapproach is that it allows the reduction of the single-substance concentrations to a minimum,thereforeenabling the use of potent,but possibly toxic,drugs.However,the optimal concentration of a singlesubstance,whether the cocktail is superior to thebest concentration of the single substances in all modalities and possible extents of interactionbetween the substances,is yet to be determined.Since results for transaminases were improved witha combination of glycine and taurine,compared tothe single substances,it can be concluded that theseresults support our hypothesis that a cocktailpotentially reduces IRI to a greater extent,comparedto the single substances.Indeed,we have not tested further combinations of all compounds included in our cocktail;however,due to the limitations of an animal model(i.e.,maximum of the reducible reperfusion injury)and the results obtained after the combination of only two compounds of the investigated cocktail,results should be sufficient to support our approach toward protection against reperfusion injury.The idea seems promising for clinical application as a further step toward achieving better outcomes in hepatic surgery.Declaration of interest:The authors report no financial conflicts of interest.The authors alone are responsible for the content and writing of this paper.REFERENCES1.Arnault I,Bao Y,Dimicoli J,Lemoine A,Sebagh M,Adam R.(2002).Combined effects of fasting and alanine on liver function recovery after cold ischemia.Transpl Int15:89Á95.2.Astarcioglu I,Adam R,Dimicoli JL,Gigou M,Patry J,Sebagh M,et al.(1995).Protective effect of alanine against graft failure of transplanted livers from fasted donor rats.Transpl Proc27:507Á508.3.Bergmeyer HU.(1988).Methods of EnzymaticAnalysis.New York:Academic Press.Cocktail to abate ischemic hepatic injury 600G Schindler et al.。