Hypothermia is better than ischemic preconditioning for preventing early hepatic ischemia/reperfusion in rats

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1 11 ORIGINAL ARTICLE Jnury-Ferury, Vol. 15 No. 1, 216: The Offiil Journl of the Mexin Assoition of Heptology, the Ltin-Amerin Assoition for Study of the Liver nd the Cndin Assoition for the Study of the Liver Hypothermi is etter thn ishemi preonditioning for preventing erly hepti ishemi/reperfusion in rts Lrisse Longo,*, Leil Xvier Siniggli-Frtt,*, Giovn R. Weer,*, Andre Jnz-Moreir,* Nélson A. Kretzmnn, Tomz de J.M. Grezzn-Filho, Norm Poss-Mrroni,* Crlos O. Corso, Crlos T. Shmidt-Cerski, Themis Reverel-d-Silveir,*, Mário R. Álvres-d-Silv,, Jorge L. dos-sntos*,, ** * Experimentl Lortory of Heptology nd Gstroenterology, Gene Therpy Center, Surgery Unit, Pthology Unit, Gstroenterology Unit, ** Peditri Heptology Unit, Hospitl de Clínis de Porto Alegre (HCPA), Porto Alegre, Brzil. Grdute Progrm in Gstroenterology nd Heptology, Universidde Federl do Rio Grnde do Sul (UFRGS), Porto Alegre, Brzil. All uthors ontriuted eqully to this study. ABSTRACT Bkground. Topil hypothermi (TH) nd ishemi preonditioning (IPC) re used to derese I/R injury. The effiy of isolted or omined use of TH nd IPC in the liver regrding inflmmtion nd ytoprotetion in erly ishemi/reperfusion (I/R) injury needs to e evluted. Mteril nd methods. Wistr rts underwent 7% liver ishemi for 9 min followed y 12 min of reperfusion. Livers of nimls lloted in the shm, normothermi ishemi (NI), IPC, TH, nd TH+IPC groups were olleted for moleulr nlyses y ELISA nd Western lot, iming to ompre proinflmmtory, nti-inflmmtory, nd ntioxidnt profiles. Re- sults. Compred with NI, TH presented deresed tumor nerosis ftor (TNF)-α, interleukin (IL)-1β, IL-6 nd IL-12 onentrtions nd inresed IL-1 levels. TH nimls displyed lower induile nitri oxide synthse (inos) nd higher endothelil nitri oxide synthse (enos) expressions. NAD(P)H-quinone oxidoredutse-1(nqo1) expression ws lso lower with TH. Isolted IPC nd NI were similr regrding ll these mrkers. TH+IPC ws ssoited with deresed IL-12 onentrtion nd redued inos nd NQO1 expressions, similrly to isolted TH. Expression of Kelh-like ECH-ssoited protein (Kep)-1 ws inresed nd expression of nuler nd ytosoli nuler erythroid 2-relted ftor 2 (Nrf2) ws deresed with TH+IPC vs. NI. Conlusion. TH ws the most effetive method of protetion ginst erly I/R injury. Isolted IPC entiled triggering of seond-line ntioxidnt defense enzymes. Comined TH+IPC seemed to onfer no dditionl dvntge over isolted TH in reltion to the inflmmtory proess, ut hd the dvntge of ompletely void seond-line ntioxidnt defense enzymes. Key words. Antioxidnt. Inflmmtion. Liver. Pthophysiology. INTRODUCTION The reversiility of ishemi-relted liver dysfuntion depends on the use, intensity, nd durtion of the ishemi proess. 1,2 Prdoxilly, even though reperfusion is essentil for proteting the liver ginst irreversile dysfuntion, it is extly during this proess tht most liver injury ours. 3,4 Hepti ishemi/reperfusion (I/R) injury results from normothermi ishemi (NI) ssoited with surgil nd linil senrios suh s liver resetion, vsulr proedures, or hypovolemi shok. In liver trnsplnttion, in whih the liver is sumitted to oth NI nd hypothermi ishemi (during storge in preservtion solution), I/R injury ounts for 1-3% of primry grft dysfuntion. 2,4,5 Vrious mehnisms ply role in I/R. 2,4,6 During reperfusion, endothelil ell edem, vsoonstrition, leuoyte dhesion, nd pltelet ggregtion in hepti sinusoids use n uneven derese in mirovsulr lood flow, produing sttered hypoxi regions. 2,4 Kupffer ells (KC) nd neutrophils re thus tivted nd serete inflmmtory meditors nd retive oxygen speies (ROS), inresing tissue dmge. 7,8 Inflmmtory meditors relted to I/R inlude, mong severl others, tumor nerosis ftor-α (TNF-α), interleukin (IL)-1β, IL-6, nd IL-12, while IL-1 ts s n nti-inflmmtory ytokine. 3,8,9 Ni- Mnusript reeived: Jnury 2, 215. Mnusript epted: April 26, 215. DOI:.1.564/

2 Prevention of ishemi/reperfusion injury., 216; 15 (1): tri oxide (NO), in ddition to induing vsodiltion, produes retive nitrogen speies (RNS), ontriuting to oxidtive stress. 1,11 An imlne etween endothelil nitri oxide synthse (enos) nd induile nitri oxide synthse (inos) promotes I/R injury If I/R injury is not soon hlted, the liver will unlesh the prodution of seond-line ntioxidnt enzymes, inluding NAD(P)H quinone oxidoredutse-1 (NQO1), y regulting signling moleules suh s the nuler erythroid 2-relted ftor 2 (Nrf2) Ativted Nrf2 is unoupled from ytosoli omplex with Kelh-like ECH-ssoited protein (Kep)-1, nd trnslotes to the nuleus, in order to ffet I/R. 6,15,17 Some therpeuti mesures hve een developed with the im of proteting the liver from I/R injury, suh s indution of topil hypothermi (TH) nd ishemi preonditioning (IPC), whih onsists in the use of rief periods of ishemi interspersed with reperfusion intervls Our group hs previously evluted the effiy of TH in isoltion or ssoited with IPC (TH+IPC) to meliorte erly stge I/R injury in rts sumitted to 9-min 7% liver ishemi followed y 12 min reperfusion. 22 In tht study, ll nimls presented ile flow lokde during ishemi; however, ile flow ws ompletely restored t 45 min of reperfusion in the groups sumitted to TH lone nd TH + IPC, ut not in the nimls sumitted to isolted IPC or mintined in NI. This suggests tht TH itself might meliorte the I/R injury, with the possiility of n dditive protetive role IPC when the two mesures were omined. The present study imed to evlute the ehvior of pro- nd nti-inflmmtory ytokines, s well s oxidtive stress nd seond-line ntioxidnt mrkers, in livers of the nimls nlyzed in tht previous experiment, helping to define t the moleulr level the mode of tion nd effiy of eh protetive method. 22 MATERIAL AND METHODS Surgil proedure This study evluted liver smples otined following euthnsi from nimls (n = 32) sumitted to I/R injury y Grezzn Filho, et l. 22 Mle Wistr rts, weighing etween 2-25 g, underwent 9 min 7% liver ishemi, inluding left nd medin loes y lmping the hepti rtery nd the portl vein nd preserving the pteny of ile duts, followed y 12 min of reperfusion. Five groups were studied: Shm (n = 4). NI (n = 7). IPC (n = 7). TH (n = 7), nd TH + IPC (n = 7). IPC onsisted of onseutive 1-min periods of ishemi nd reperfusion efore the ishemi insult. TH ws indued y the superfusion of ooled sline t 26 C onto the ishemi loes. All nimls were euthnized immeditely fter the end of the experiment. Smples were immeditely stored in liquid nitrogen nd kept t -8 C. Protein onentrtion ssessment Protein onentrtion in tissue homogentes ws ssessed in ordne with the tehnique desried y Brdford. 23 Smples were nlyzed y spetrophotometry t 595 nm nd the otined vlues were expressed in mg/ ml. These vlues were employed for the lultions of ELISA nd Western lot ssys. For eh method, speifi homogentes were prepred. Enzyme linked immune sorent ssy (ELISA) ELISA ws used to ssess onentrtions of IL-1β (ebiosiene 88-61, Sn Diego, CA, USA), TNF-α (ebiosiene , Sn Diego, CA, USA), IL-6 (BioSoure KRC61, Cmrillo, CA, USA), IL-12p7 (Bio- Soure KRC2371, Cmrillo, CA, USA), nd IL-1 (BioSoure KRC12, Cmrillo, CA, USA). The liver tissue smples were homogenized in PBS uffer ontining protese inhiitor mix/oktil (Sigm P834, Sint Louis, MO, USA) in ie nd entrifuged t 4, rpm for 1 min t 4 C. The superntnt ws olleted for determining the onentrtion of the studied mrkers. All nlyses were done ording to mnufturer instrutions. Asorne ws mesured y spetrophotometer t 45 nm. Color intensity ws diretly proportionl to ytokine onentrtion in the smples. Results were expressed in pg/mg of protein. Cytoplsm nd nuler extrts preprtion Cytoplsm extrts for determining enos, inos, Nrf2, Kep1 nd NQO1 protein expressions, s well s nuler extrts for sertining nuler Nrf2 protein expressions, ll y Western lot, were prepred from liver tissue frgments using protool dpted from Sdowiski & Gilmn. 24 Briefly, liver smples were homogenized in ie with 5 μl hypotoni uffer A (1M HEPES ph 7.9,.5M NF, 1 mm N3VO4, 1 mm glyerolphosphte N,.5M EDTA-N ph 7.5, 1 mm EGTA ph 7.5 nd 1mM DTT). Nonidet P-4 nd protese inhiitor were

3 112 dded to this solution. Smples were entrifuged t 1, rpm during 1 min t 4 C. The superntnt ws removed nd stored t -8 C for Western lotting of the ytoplsmi extrt. The nuler pellet present in the liver sediment frtion ws resuspended with 5 μl of hypotoni uffer B (1M HEPES ph 7.9,.5M NF, 1mM N 3 VO 4, 4M NCl, 1 mm glyerolphosphte N,.5M EDTA- N ph 7.5, 1 mm EGTA ph 7.5 nd 1 mm DTT) nd vortexed t 5 min intervls during 3 min. Nuler extrts were then entrifuged t 12, rpm during 2 min t 4 C nd the superntnt ws stored t -8 C until the omplishment of the Western lot ssy. Western lot Smples ontining 1 μg of protein were seprted y sodium dodeyl sulfte-polyrylmide gel eletrophoresis (9-12% rylmide) nd trnsferred to polyvinylidene fluoride memrnes. The memrnes were then loked with 5% nonft dry milk in PBS ontining.5% Tween 2 (PBS-T) for 1 h t room temperture nd proed overnight t 4 C with polylonl nti-enos (SC 8311/ 13 kd), nti-kep1 (SC 33569/ 7 kd), nti- Nrf2 (SC 3915/ 57 kd), monolonl nti-inos (SC 7271/ 12 kd), nti-nqo1 (SC 37623/ 31 kd) nd nti-β-tin (SC 8432/ 43 kd) ntiodies (Snt Cruz Biotehnology, Snt Cruz, CA, USA) t 1:2-1:1, dilution with PBS-T in 5% nonft dry milk. After the memrnes were wshed with PBS-T nd inuted for 1 h t room temperture with seondry HRP-onjugted ntiody (Snt Cruz Biotehnology, Snt Cruz, CA, USA) t 1:5,-1:1, dilution with PBS-T in 5% nonft dry milk. Protein detetion ws performed vi hemiluminesene using ommeril ECL kit (WBKLS5 Millipore Co., Billeri, MA, USA). The density of the speifi nds ws quntified with n L-Pix Chemi Moleulr Imging densitometer. Results were expressed in ritrry units (u). Liver histology Smples of liver tissue from every studied niml were formlin-fixed nd emedded in prffin, nd five milimeter setions were otined nd stined with hemtoxylin nd eosin. Slides were evluted under light mirosopy with 4 x mgnifition. A semi-quntittive ssessment of the liver histopthology ltertion ws done ording to the sle developed y Suzuki, et l., whih ssigns vlue from to 4 to oserved morphologi ltertions in reltion to sinusoidl ongestion, neutrophil infiltrtion nd heptoellulr nerosis. 25 The sum of these vlues ws lulted for eh niml, nd the sores were presented s men of eh group. The histologi evlution ws performed y one expert in Pthology, linded in regrds to the experimentl group in whih every single niml ws inluded. Sttistis The lultion of the neessry smple size ws sed on the preliminry nlysis of the ehvior of the pro-inflmmtory moleules TNF-α nd IL-1β evluted y the ELISA ssy. We onluded tht the numer of 32 nimls, previously used in the study y Grezzn Filho, et l. onferred sttistil power of 8% t 5% level signifine to the present study. 22 Normlity of dt distriution ws evluted using the Shpiro-Wilk test. Beuse distriutions were prmetri in the omprison etween groups, ANOVA ws used followed y the Tukey post-ho test. P <.5 ws onsidered sttistilly signifint. Ethis The study ws pproved y the Ethis Committee t Hospitl de Clínis de Porto Alegre, whih follows the Counil for Interntionl Orgniztion of Medil Sienes (CIOMS) ethil ode for niml experimenttion. RESULTS In this study, the NI group represented the unproteted liver ginst I/R injury, while shm nimls were employed s norml ontrols. Animls sumitted to the protetive methods IPC, TH nd TH + IPC were ompred with oth these groups. Effet of protetive methods on inflmmtory ytokine ehvior We sought to determine the effets of eh protetive method on erly I/R injury in the liver refleted y the onentrtion of inflmmtory moleules (Figures 1A- 1D). Signifint differenes were oserved mong the groups for the onentrtions of ll inflmmtory moleules evluted: TNF-α (P <.1), IL-1β (P =.1), IL-6 (P <.1), IL-12 (P <.1). The onentrtions of ll these moleules were deresed in the TH group in omprison to NI: TNF-α (P <.1), IL-1β (P =.5), IL-6 (P =.1) nd IL-12 (P =.2), with vlues similr to those of the shm group. The IPC nd TH + IPC groups were similr to NI for TNF-α (P =.416 nd P =.873, respetively), IL-1β (P =.659 nd P =.419 respetively), IL-6 (P =.997 nd P =.149 respetively). IL-12 onentrtion ws deresed in the TH + IPC group in omprison to the NI group (P =.16), with

4 Prevention of ishemi/reperfusion injury., 216; 15 (1): vlues omprle to the shm group, while the IPC group presented IL-12 onentrtions similr to those of NI (P =.411). Effet of protetive methods on nti-inflmmtory ytokine IL-1 We evluted the role of nti-inflmmtory ytokine IL-1 in the protetion ginst I/R injury. Results re desried in figure 2. Liver onentrtions of IL-1 were signifintly different mong the groups (P <.1), with the TH group presenting higher IL-1 onentrtions vs. the NI group (P =.15). In the isolted IPC nd TH + IPC groups, IL-1 levels were similr to those of the NI group (P =.988 nd P =.981 respetively). Effet of protetive methods on nitri oxide synthse isoforms In ddition to inflmmtion mrkers, we investigted the expression of nitri oxide synthses with eh protetive method (Figures 3A-3C). enos expression differed signifintly mong the groups (P =.6), with the TH group displying higher expression in omprison with oth the NI (P =.37) nd TH + IPC (P =.6) groups. Regrding inos expression, signifint differenes were lso oserved mong the groups (P <.1). The shm group presented the lowest expression ompred to NI (P <.1), IPC (P =.13), nd TH + IPC (P =.29), while TH nd TH + IPC presented lower expression in omprison with oth NI (P =.1 nd P <.1, respetively) nd isolted IPC (P =.34 nd P =.13 respetively). Ativtion of the seond-line ntioxidnt defenses y protetive methods We tried to identify whih protetive method tivted seond-line ntioxidnt defenses. For tht, the expression of NQO1 protein nd the ehvior of the Kep1-Nrf2 omplex, inluding ytosoli nd nuler Nrf2 expression, were nlyzed (Figures 4A-4F). We oserved differenes mong groups in the expression of NQO1 (P =.1), Kep1 (P =.27), nd ytosoli (P =.6) nd A 2, 1,5, B 5, 4,,, TNF-α (pg/mg) 1, 5 IL-1β (pg/mg) 3, 2, 1, C Shm NI IPC TH TH + IPC Shm NI IPC TH TH + IPC D IL-6 (pg/mg) 8, 6, 4, 2,, IL-12 p7 (pg/mg) 2, 15, 1, 5,, Shm NI IPC TH TH + IPC Shm NI IPC TH TH + IPC Figure 1. Liver expression of TNF-α (A), IL-1β (B), IL-6 (C) nd IL-12 p7 (D) fter reperfusion (y ELISA ssy). Dt expressed s men ± SEM. Different letters indite signifint differene oserved in the mens of groups (P <.5). NI: normothermi ishemi. IPC: ishemi preonditioning. TH: topil hypothermi.

5 114 IL-1 (pg/mg) 4, 3, 2, 1,, A enos inos β-tin Shm NI IPC TH TH + IPC Shm NI IPC TH TH + IPC Figure 2. Liver expression of IL-1 fter reperfusion (y ELISA ssy). Dt expressed s men ± SEM. Different letters indite signifint differene oserved in the mens of groups (P <.5). NI: normothermi ishemi. IPC: ishemi preonditioning. TH: topil hypothermi. B enos/β-tin (ritrry units) nuler (P =.5) Nrf2. NQO1 expression ws similr in the IPC nd NI (P =.991) groups, nd ws higher in oth these groups s ompred to the shm (P =.22 nd P =.42, respetively), TH (P =.9 nd P =.2, respetively) nd TH + IPC (P =.1 nd P =.22, respetively) groups. Kep1 protein expression in TH + IPC group ws higher thn in NI (P =.35). The NI group displyed the highest ytosoli Nrf2 expression, with vlues similr to those oserved in the isolted IPC group (P =.722), nd the highest nuler Nrf2 expression, whih ws higher thn tht reorded in the shm (P =.4) nd TH + IPC (P =.29) groups. Effet of protetive methods on histopthologil sore The histopthology effet of the I/R injury fter 2-h reperfusion period ws evluted (Figures 5A-5F). The overll omprison mong ll the groups showed signifint differenes in reltion to the histopthology sore (P =.11). Regrding the differenes etween the speifi groups, signifint differene ws oserved etween the NI nd shm groups (P =.12). The remining groups presented only negligile ltertions. DISCUSSION The mehnisms y whih TH nd IPC operte in I/R injury re not ompletely ler, ut they re thought to ply ruil role in relieving inflmmtion nd prodution of ROS nd RNS following reperfusion. 18,19,21,26 The ssoition of TH + IPC hs een used in rdi nd retinl surgeries Regrding the liver, our group ws the first to report on the effetiveness of this synergisti pproh C inos/β-tin (ritrry units) Shm NI IPC TH TH + IPC Shm NI IPC TH TH + IPC Figure 3. Liver expression of nitri oxide synthse isoforms. A. Western lot nlysis. B. enos expression (densitometry nlysis). C. inos expression (densitometry nlysis). Dt expressed s men ± SEM. Different letters indite signifint differene oserved in the mens of groups (P <.5). NI: normothermi ishemi. IPC: ishemi preonditioning. TH: topil hypothermi. to prevent erly I/R injury in rt model. 22 We nlyzed the protetive effet of isolted or omined TH nd IPC on ile flow nd on the ehvior of first-line ntioxidnt enzymes, ompring these tretment groups with shm nd NI groups. We oserved tht in nimls sumitted to TH nd TH + IPC, ile flow ws restored during reperfusion fter ile flow lokde during ishemi. The present study ws designed to ontinue tht initil investigtion. For tht, we foused on the moleulr mehnisms nd effetiveness

6 Prevention of ishemi/reperfusion injury., 216; 15 (1): A NQO1 B.8.6 Kep 1 Cytosoli Nrf2 β-tin NQO1/β-tin (ritrry units).4.2 Shm NI IPC TH TH + IPC. Shm NI IPC TH TH + IPC C D Kep1/β-tin (ritrry units) Cytosoli Nrf2/β-tin (ritrry units) Shm NI IPC TH TH + IPC. Shm NI IPC TH TH + IPC E F 2. Nuler Nrf2 β-tin Nuler Nrf2/β-tin (ritrry units) Shm NI IPC TH TH + IPC. Shm NI IPC TH TH + IPC Figure 4. Liver expression of NQO1, Kep1 nd Nrf2 (ytosoli nd nuler). A. Western lot nlysis. B. NQO1 expression (densitometry nlysis). C. Kep1 expression (densitometry nlysis). D. Cytosoli Nrf2 expression (densitometry nlysis). E. Western lot nlysis. F. Nuler Nrf2 (densitometry nlysis). Dt expressed s men ± SEM. Different letters indite signifint differene oserved in the mens of groups (P <.5). NI: normothermi ishemi. IPC: ishemi preonditioning. TH: topil hypothermi.

7 116 of TH nd IPC methods used lone or in omintion for proteting ginst hepti I/R injury, onsidering pro- nd nti-inflmmtory moleules s well s mrkers ssoited with the seond-line ntioxidnt defense. 22 Hepti I/R injury n e lssified into two stges in reltion to reperfusion: n erly phse, orresponding to the first 6 h fter the strt of reperfusion, nd lte phse tht lsts 48 h. 5,12 In erly I/R, tivted KC serete pro-inflmmtory meditors, suh s TNF-α, IL-1β, nd IL-6, whih intensify ell injury. 3,7 In this study, the TH group showed deresed hepti onentrtions of TNF-α nd IL-1β in omprison with the NI group, nd the onentrtion of these moleules in the TH group ws similr to tht found in shm nimls. In the presene of I/R injury, TNF-α nd IL-1β hve similr tivity, exerting stimultory effet on the expression of other inflmmtory ytokines nd hemokines, regulting free rdil prodution, nd helping neutrophil reruitment nd dhesion to sinusoidl endothelil ells. 3 The present findings gree with those otined y other groups, showing tht the use of hypothermi in hepti I/R injury redues inflmmtion y lowering TNF-α nd IL- 1β prodution. 18,3,31 Mhmoud, et l. oserved tht inhiition of TNF-α indues deresed inflmmtion nd oxidtive stress, possily providing useful therpeuti pproh to prevent I/R injury. 8 In the present study, TNF-α nd IL-1β onentrtions were similr in IPC nd NI nimls. Koneru, et l. did not oserve dvntges in using IPC during surgil proedures to derese TNF-α onentrtions. 32 In this study, hepti IL-6 onentrtions were deresed in TH nd shm groups in omprison with NI, while in the IPC group IL-6 onentrtions were not different from NI. IL-6 is n importnt mrker of tissue dmge nd inflmmtion, nd deresing its expression minimizes I/R injury. 33 The present findings suggest tht TH, nd not IPC, hs protetive role ginst hepti inflmmtion. Qi, et l. using n niml model of heptoellulr rinom, reported tht the use of IPC indued dereses in TNF-α nd IL-1β serum levels, ssoited with inresed levels of IL-6 nd higher signl trnsdution nd tivtion of trnsription 3 (STAT3) expression. 18 They proposed tht the enefiil effets of IPC might e nelled out y the tivtion of the IL-6/ STAT3 pthwy, thus elerting rinogenesis. Other studies, however, propose tht inresed IL-6 onentrtions led to STAT3 tivtion, induing the expression of mitogeni nd nti-poptoti proteins ple of proteting ginst I/R injury nd ontriuting to heptoellulr regenertion. 34,35 In the present study, TH, lone or ssoited with IPC, led to diminished hepti IL-12 onentrtion, with levels similr to those of the shm group. IL-12 is pro-inflmmtory meditor involved in hepti I/R injury, nd niml models hve reveled tht exogenous pplition of this ytokine led to KC tivtion, expression of ell dhesion moleules, nd neutrophil umultion in liver prenhym. Conversely, mie treted with neutrlizing ntiodies for IL-12 presented deresed TNF-α nd interferon-γ expression, using redued neutrophil reruitment to liver prenhym. 9,36 In the present study, the IPC group presented the highest onentrtions of IL-12, suggesting tht IPC does not protet ginst the tion of the pro-inflmmtory meditors tht worsen hepti I/R injury. In turn, IL-1 (previously lled ytokine synthesis inhiitory ftor) plys n importnt nti-inflmmtory role ginst I/R injury. In this study, not only ws TH responsile for deresing the onentrtion of inflmmtory ytokines, it lso used inresed seretion of IL-1 in omprison with the NI group. IL-1 levels were similr in livers of IPC nd NI nimls. This finding differs from the report y Serfín, et l. who oserved protetive effet of IPC in livers of nimls with ftty liver disese, ssoited with inresed levels of IL-1 nd redued inflmmtion. 37 Some studies hve reported loked nuler ftor-κβ tivity s result of IL-1 seretion in I/R injury, nd thus the expression of inflmmtory meditors, proteting liver ginst ell dmge. 3,7,38 NO plys key role in the regultion of liver lood flow in norml onditions, ut the inresed prodution of NO during I/R injury ggrvtes hepti tissue dmge owing to free rdil prodution. 1,12 NO is synthesized through the tion of NOS. enos leds to the prodution of physiologil mounts of NO, while inos prompts opious nd lsting NO synthesis, thus explining inos involvement in severl pthologi onditions. 1,11 The present study showed higher enos expression in the TH group ompred to NI. NO synthesis ttriutle to the tion of enos hs protetive effet ginst I/R injury, modulting vsodiltion nd inhiiting the prodution of inflmmtory ytokines nd free rdils y KC. 12,39,4 In this study, higher inos expression ws lso oserved in the IPC group s ompred to the TH lone or TH+IPC group, ut similr to the NI group. inos derived NO my e toxi or protetive, depending on the type of ggression, on NO levels, nd on the durtion of inos expression. 4,11 Our findings indite tht TH provided protetion ginst I/R injury in liver y promoting n inrese in the levels of enos, while IPC, on the ontrry, led to inresed expression of inos, whih my ggrvte heptoellulr dmge. ROS nd RNS re generted long the entire period of I/R injury, ut the detrimentl effet of oxidnt gents ssoited with derese in ntioxidnt defenses produes oxidtive stress. 2,4 The ntioxidnt enzymes superoxide

8 Prevention of ishemi/reperfusion injury., 216; 15 (1): A B C D E F 6 Histopthologil sore 4 2 Shm NI IPC TH TH + IPC Figure 5. Liver histology of the groups under study nd omprison y the histopthologil sore. Slide imges refer to shm (A), NI (B), IPC (C), TH (D), TH+IPC (E) groups. The NI group presented sinusoidl ongestion, neutrophil infiltrtion nd heptoellulr nerosis, ltertions not found in the remining groups. Mgnifitions: 4X. 5f- Comprison y the histopthologil sore. Dt expressed s men ± SEM. Different letters indite signifint differene oserved in the mens of groups (P <.5). NI: normothermi ishemi. IPC: ishemi preonditioning. TH: topil hypothermi.

9 118 dismutse (SOD), tlse nd glutthione peroxidse re lssified s first-line ntioxidnt defenses ginst I/R injury. 41 Grezzn Filho, et l. showed tht TH indued inresed levels of SOD in omprison with NI, IPC nd TH+IPC. 22 The inresed levels of SOD indued y TH re ple of olishing the dmge used y I/R injury, preluding the use of seond-line ntioxidnt defenses. Seond-line ntioxidnt defense enzymes re tivted whenever those in the first line fil. 17,41 Seond-line enzymes synthesis is medited through tivtion of the Nrf2-ntioxidnt response element signling pthwy. 6,13 In norml onditions, Nrf2 is loted in ytoplsm, ssoited with its repressor Kep1, oth forming n intive omplex. Under stress ondition, induers suh s ROS oxidize Kep1 ysteine residue, leding to dissoition of the inhiitory omplex. 15,17 Nrf2 unouples from Kep1, nd trnslotes to the nuleus in order to promote the gene trnsription of seond-line ntioxidnt enzymes, suh s NQO However, during short time spn efore entering the nuleus, tivted Nrf2 umultes in ytoplsm. 17 In this study, TH ws effetive in ontrolling the genertion of ROS, proteting ginst the ellulr dmge used y reperfusion. The TH group hd the lowest onentrtions of inflmmtory ytokines nd the highest IL-1 levels, s well s more fvorle liver vsotive response s ssessed y inos nd enos expression. These findings indite lower prodution of ROS nd RNS nd n dequte restortion of the ell redox lne with TH. Redued oxidtive stress prevents dmge to lipids, proteins, nd DNA. 2,16 The use of hypothermi requires speil re in terms of hemodynmi monitoring, sine it n trigger some deleterious effets suh s edem, ltte umultion nd intrellulr idosis. 21,27 Severl methods of hypothermi pplition hve desried, inluding the TH. Ymnk, et l. studied ptients with hroni liver diseses nd heptoellulr rinom who underwent right-sided segmentetomy under normothermi or TH with tempertures rnging from 2 to 25 C. 42 Those uthors showed tht the ptients sumitted to TH presented redued lood loss nd tolerted lrger ishemi period in omprison with those who underwent the proedure under normothermi. Benefiil effets of moderte hypothermi hve lso een reported in studies tht evluted ptients suffering from ute liver filure. 43 Conversely, NQO1 expression ws higher in the IPC group in omprison with the TH nd TH+IPC groups, suggesting tht IPC filed to ontrol oxidtive stress through first-line ntioxidnt defense enzymes. Shokeir, et l. evluted Nrf2 nd NQO1 expressions in nimls sumitted to the IPC pproh ginst renl I/R injury. 13 The results otined y those uthors re similr to those of our study, with IPC nimls presenting higher levels of Nrf2 nd NQO1 expression in omprison with the unproteted smple. Some studies, however, report synergi effet of NQO1 nd SOD, whih protets ells ginst the genertion of ROS nd RNS. 16,44 In the present study, TH lone or in omintion with IPC produed the lowest expression of Nrf2 in ytoplsm, inditing less Nrf2- Kep1 unoupling nd tivtion nd suggesting tht in this group tivtion of the seond-line defense system ws unneessry. Isolted IPC, on the ontrry, produed similr ytosoli Nrf2 expression to NI, inditing filure of the first ntioxidnt defense line. The omintion of the two methods, TH nd IPC, used omplete inhiition of Kep1/Nrf2 unoupling, preventing trnslotion of Nrf2 to the nuleus. This strong inhiition resulted in redued expression of oth NQO1 nd nuler Nrf2. These findings suggest tht the ssoition of IPC with TH my ontriute n dditionl protetive effet s ompred to TH lone. In this study, findings of signifint histopthologi injury ourred only in the NI group in omprison with shm group. Dinnt, et l. evluted the protetive effets of ooling y in situ hypothermi perfusion with Ringer solution t 4 C during 6-min of totl vsulr exlusion in porine liver I/R model. 45 Those investigtors showed signifint inrese of histopthologi injury in the normothermi group, whih presented inresed liver ell vuoliztion, neutrophil infiltrtion nd prenhyml nerosis. The histopthologi ltertions were not oserved in the nimls sumitted to hypothermi. The histopthologi ltertions nd d outome were not oserved in the nimls sumitted to hypothermi. Nieuwenhuijs, et l. evluted the ile flow nd histologi ltertions in n experimentl protool of IPC, using 1-min ishemi nd 1-min reperfusion periods followed y 6-min reperfusion phse. 46 Those uthors oserved tht the ile flow levels otined t reperfusion were dependent on the length of the ishemi period. Signifint histopthologi ltertions ssoited to the funtionl disturne were sent sine, ording to the uthors, nerosis nd poptosis our only in the lte phses of reperfusion. One unexpeted finding in this study ws tht isolted IPC ws ineffetive ginst I/R injury in the liver. Although most studies present evidenes fvoring the use of IPC ginst the I/R injury in the liver, some groups showed tht this method offers only minor enefiil effets, or is useless. 47,48 Psupthy, et l. reported tht IPC offers protetive effet ginst I/R injury in two distint time points: in the erly stge, omprising the first 3 h fter the orgn reperfusion, nd in the lte stge, etween 12 nd 24 h. If IPC is pplied in this lst sitution, the protetive effet n persist for up to 3 dys. 49 A puttive explntion for our divergent findings is tht the 9 min ishemi

10 Prevention of ishemi/reperfusion injury., 216; 15 (1): hosen in the design of this study n e eyond the limits of the protetion ffordle y IPC. Additionlly, other groups otined similr results to our findings. 47,5 Presently, our tem is rrying out projets mking use of the sme experimentl model, ut pplying longer period of reperfusion, nd ssessing histologi nd liver enzyme ltertions in the lte phse of reperfusion (24 h). In onlusion, using n niml model of erly I/R injury, we showed tht TH lone ws more effetive thn IPC lone to protet the liver ginst inflmmtion nd oxidtive stress, preluding the need to tivte seond-line ntioxidnt defenses. Isolted IPC did not hve protetive effet s indited y the mrkers evluted in this study. ABBREVIATIONS enos: endothelil nitri oxide synthse. I/R: ishemi/reperfusion. IL: interleukin. inos: induile nitri oxide synthse. IPC: ishemi preonditioning. KC: Kupffer ells. Kep1: Kelh-like ECH-ssoited protein-1. NI: normothermi ishemi. NO: nitri oxide. NQO1: NAD (P)H quinone oxidoredutse-1. Nrf2: nuler erythroid 2-relted ftor 2. RNS: retive nitrogen speies. ROS: retive oxygen speies. SOD: superoxide dismutse. TH: topil hypothermi. TNF-α: tumor nerosis ftor-α. GRANT SUPPORT Fundo de Inentivo Pesquis e Eventos-Hospitl de Clínis de Porto Alegre (FIPE-HCPA) nd Coordenção de Aperfeiçomento de Pessol de Nível Superior (CAPES). REFERENCES 1. Knudsen AR, Knnerup AS, Grønæk H, Dutoit SH, Nyengrd JR, Funh-Jensen P, Mortensen FV. Quntittive histologil ssessment of hepti ishemi-reperfusion injuries following ishemi pre- nd post-onditioning in the rt liver. J Surg Res 213; 18: Ildefonso JA, Aris-Díz J. Pthophysiology of liver ishemi-reperfusion injury. Cir Esp 21; 87: Dinnt S, Veteläinen RL, Florquin S, vn Vliet AK, vn Gulik TM. IL-1 ttenutes hepti I/R injury nd promotes heptoyte prolifertion. J Surg Res 27; 141: Glntzounis GK, Slinski HJ, Yng W, Dvidson BR, Seiflin AM. The ontemporry role of ntioxidnt therpy in ttenuting liver ishemi-reperfusion injury: review. Liver Trnspl 25; 11: Teoh NC. Hepti ishemi reperfusion injury: Contemporry perspetives on pthogeni mehnisms nd sis for heptoprotetion-the good, d nd dedly. J Gstroenterol Heptol 211; 26: Ke B, Shen XD, Zhng Y, Ji H, Go F, Yue S, Kmo N, et l. KEAP1-NRF2 omplex in ishemi-indued heptoellulr dmge of mouse liver trnsplnts. J Heptol 213; 59: Kireev RA, Cuest S, Irrol C, Bel T, Moreno Gonzlez E, Vr E, Tresguerres JA. Age-relted differenes in hepti ishemi/reperfusion: gene tivtion, liver injury, nd protetive effet of meltonin. J Surg Res 212; 178: Mhmoud MF, El Shzly SM, Brkt W. Inhiition of TNF-α protets ginst hepti ishemi-reperfusion injury in rts vi NF-κβ dependent pthwy. Nunyn Shmiedeergs. Arh Phrmol 212; 385: Lentsh AB, Yoshidome H, Kto A, Wrner RL, Chedle WG, Wrd PA, Edwrds MJ. Requirement for interleukin-12 in the pthogenesis of wrm hepti ishemi/reperfusion injury in mie. Heptology 1999; 3: Miyke T, Yokoym Y, Kokuryo T, Mizutni T, Immur A, Ngino M. Endothelil nitri oxide synthse plys min role in produing nitri oxide in the superute phse of hepti ishemi prior to the upregultion of induile nitri oxide synthse. J Surg Res 213; 183: Tsuhihshi S, Klds F, Chid N, Sudo Y, Tmur K, Zhi Y, Qio B, et l. FK33, novel induile nitri oxide synthse inhiitor, prevents ishemi nd reperfusion injury in rt liver trnsplnttion. Am J Trnsplnt 26; 6: Hines IN, Hrd H, Flores S, Go B, MCord JM, Grishm MB. Endothelil nitri oxide synthse protets the postishemi liver: potentil intertions with superoxide. Biomed Phrmother 25; 59: Shokeir AA, Hussein AM, Brkt N, Adelziz A, Elgr M, Awdll A. Ativtion of nuler ftor erythroid 2-relted ftor 2 (Nrf2) nd Nrf-2-dependent genes y ishemi pre-onditioning nd post-onditioning: new dptive endogenous protetive responses ginst renl ishemi/reperfusion injury. At Physiol (Oxf) 213; 21: Kudoh K, Uhinmi H, Yoshiok M, Seki E, Ymmoto Y. Nrf2 tivtion protets the liver from ishemi/reperfusion injury in mie. Ann Surg 214; 26: Leonrd MO, Kiern NE, Howell K, Burne MJ, Vrdrjn R, Dhkshinmoorthy S, Porter AG, et l. Reoxygention-speifi tivtion of the ntioxidnt trnsription ftor Nrf2 medites ytoprotetive gene expression in ishemi-reperfusion injury. FASEB J 26; 2: Gng GT, Hwng JH, Kim YH, Noh JR, Kim KS, Jeong JY, Choi DE, et l. Protetion of NAD(P)H:quinone oxidoredutse 1 ginst renl ishemi/reperfusion injury in mie. Free Rdi Biol Med 214; 67: Morles P, Vrgs R, Videl LA, Fernández V. Nrf2 tivtion in the liver of rts sujeted to preonditioning suhroni iron protool. Food Funt 214; 5: Qi Q, Bie P. Different roles of hepti hypothermi ishemi nd ishemi preonditioning in hemilly indued heptorinogenesis in rts. J Surg Res 214; 189: Behrends M, Hirose R, Serkov NJ, Cotney JL, Bedolli M, Yrdi J, Prk YH, et l. Mild hypothermi redues the inflmmtory response nd hepti ishemi/reperfusion injury in rts. Liver Int 26; 26: Clvien PA, Selzner M, Rüdiger HA, Grf R, Kdry Z, Rousson V, Johum W. A prospetive rndomized study in 1 onseutive ptients undergoing mjor liver resetion with

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Lortório Experimentl de Heptologi e Gstroenterologi, Centro de Pesquis Experimentl do Hospitl de Clínis de Porto Alegre. Ru Rmiro Brelos, 235/ sl CEP 935-3, Birro Rio Brno, Porto Alegre, RS, Brzil. Ph.: Fx: E-mil: lrisselongo@hotmil.om