Hormonal pretreatment preserves liver regenerative capacity and minimizes inflammation after partial hepatectomy

Transcription

1 ORIGINAL ARTICLE Hormonl pretretment fter prtil heptectomy., 213; 12 (6): November-December, Vol. 12 No.6, 213: Hormonl pretretment preserves liver regenertive cpcity nd minimizes inflmmtion fter prtil heptectomy Amnd D Espessilles,* Cmil Dossi,** Giomr Intrigo,** Pedro Leiv,** Pmel Romnque**, *** * Fcultd de Ciencis, ** Instituto de Ciencis Biomédics, Fcultd de Medicin, Universidd de Chile; *** Fcultd de Medicin, Universidd Diego Portles. ABSTRACT Introduction. The tretment of brin ded donors with combined hormonl resuscittion protocols, including methylprednisolone (MP) nd triiodothyronine (T3), mong others, ws developed to increse the vibility nd function of trnsplnted orgns, primrily hert nd lung. Even when it hs regrded successful results in term of donors nd orgns recovery, its effects over specific prmeters in orgns like the liver re unknown. Mteril nd methods. Mle Sprgue-Dwley rts were pretreted with MP (.34 mg/kg) nd/or T3 (.5 mg/kg) or their vehicles, nd then subjected to prtil heptectomy of 7%. Three experimentl groups nd their respective controls were conformed:. T3; b. NOH; c. MP; d. vmp; e. MP+T3 nd f. vmp+noh. The groups were evluted t, 16, 24, 72 nd 12 h post surgery. The effects of this protocol on regenertion, liver mss recovery, liver injury, oxidtive stress nd liver function were nlyzed. Results. MP+T3 pretretment does not deleteriously ffect liver regenertion fter prtil heptectomy, s shown in the curve of totl mss recovery, Ki67 stining nd mitosis counting, nd does not lter liver function. In ddition, the tretment modestly decreses oxidtive stress nd liver injury, s evidenced by trnsminses levels, histologicl nlysis nd oxidized proteins content. Conclusion. These preclinicl results indicte tht MP+T3 is hrmless for liver tissue regenertion post heptectomy nd dditionlly exhibits nti-inflmmtory nd ntioxidnt effects; therefore, it would not be contrindicted for the tretment of multiorgn donors in brin deth nd prticulrly, if the occurrence of smll for size syndrome is suspected. Key words. Hormonl resuscittion protocol. Liver regenertion. Brin deth donor. INTRODUCTION Success of orgn trnsplnttion is dependent on severl fctors, mong them, orgn qulity. Brin deth (BD) is the condition of most donors during trnsplnttion, nd is ccompnied by hemodynmic, hormonl nd inflmmtory ltertions, which cn irreversibly dmge orgn function. 1 To minimize the occurrence of these events, severl strtegies hve been developed; one of them, hormonl resuscittion, is becoming more frequently used nd hs Correspondence nd reprint request: Pmel Romnque Instituto de Ciencis Biomédics, Fcultd de Medicin, Universidd de Chile. Independenci 127, Zóclo D, Independenci, Sntigo, Chile. Tel: E-mil: promnque@med.uchile.cl Mnuscript received: April 1, 213. Mnuscript ccepted: My 13, 213. proven to increse the qulity of extrcted orgns, s well s recovery of donors nd grfts. 2 Hormonl therpy, lso known s T4 protocol or hormonl resuscittion protocol, my include the use of thyroid hormones, vsopressin, insulin nd corticoids. It ws initilly pplied by Rosendle nd Novitzky s group with successful results, 2 prticulrly in crdic function; 3 however, its effect over specific prmeters in other orgns, like liver nd kidney, is poorly studied. The incidence of primry grft nonfunction (PNF) is relted to ischemi-reperfusion liver injury, orgn qulity nd donor risk index. 4 The incidence of PNF in hormonl treted donors hs not been published nd some uthors suggest it my be higher. 5 Results hve been communicted regrding the role of hormonl therpy nd the detrimentl response of stetotic liver to ischemi-reperfusion injury in experimentl models; 5 nevertheless, the effect of such protocols in norml liver response to injury hs not been reported.

2 882 D Espessilles A, et l., 213; 12 (6): Orgn trnsplnttion is complex procedure nd the potentil dmge of the orgn is due to preservtion, cold nd wrm ischemi, reperfusion, nd the surgery per se. It is of the outmost importnce for the liver to preserve the prolifertion cpcity in order to recover tissue dmge. 6 Furthermore, in the cse of BD donors where the liver does not mtch the receptor size, regenertion is vitl to meet the smll for size syndrome. 7 Liver regenertion is n evolutionry conserved process tht involves the interction of liver cells, signling pthwys, prcrine signls, nd influence from other orgns. In humns, liver regenertion occurs when recovery of lost heptocytes is needed, for exmple fter liver dmge from heptitis, liver filure, ischemi-reperfusion, prtil heptectomy (PHx) nd trnsplnttion, mong others. 6 One fctor tht gretly influences outcome fter trnsplnttion is liver cpcity to regenerte fter trnsplnt, which is modified by severl conditions, including extension of ischemic injury, grft size, immunosuppression, stetosis, donor ge nd virl heptitis. 8 Our im ws to study the effect of the combined dministrtion of triiodothyronine nd methylprednisolone over liver regenertion fter prtil heptectomy, considering this cpcity s n importnt fctor for the outcome fter liver trnsplnttion. MATERIAL AND METHODS Animls nd tretments Mle Sprgue-Dwley rts (Animl Fcility, Fculty of Medicine, University of Chile) weighing 18-2 g, housed on 12-h light/drk cycle nd provided with rt chow nd wter d libitum, were used throughout the experiments. Triiodothyronine (T3,.5 mg/kg, Sigm-Aldrich, USA) nd/or methylprednisolone (MP, 34 mg/kg, Solu-medrol, Pfizer, Switzerlnd) were dministered 2 nd 3 h before surgery, respectively. Control nimls received n equivlent volume of T3 vehicle (NOH.1 N) nd/or MP vehicle (provided by the mnufcturer). Prtil heptectomy Animls were nesthetized with 2% isoflurne nd subjected to prtil heptectomy: briefly, medin, right centrl nd left lterl lobes, which constitute 7% of the totl liver, were seprtely ligted, resected nd removed under microscope. 9 Control (shm surgery) consisted in bdominl wll opening, cvity exposure nd closure. Resected liver ws weighed nd frozen in liquid nitrogen. Recovery ws llowed in controlled environment, buprenorphine hydrochloride.1 mg/kg (temgesic, 324 mcg/ml, Essex Phrm, Munchen, Germny) ws dministered, nd dily monitoring including body weight nd clinicl condition ws ssessed until hrvesting time. Liver hrvesting ws performed in nesthetized nimls (.2 ml Zoletil, Tiletilmine Clorhidrte 5 mg/kg, Zolzepm Clorhidrte 5 mg/kg, Virbc, Frnce). Remnnt liver ws weighed nd blood smples tken, centrifuged, nd serum storge t -2 C. Tissue smples were formlin-fixed nd prffin included or frozen in liquid nitrogen nd storge t -8 C for further determintions. Experimentl niml protocols nd procedures complied with the Guide for the Cre nd Use of Lbortory Animls nd pproved by locl uthorities (Fculty of Medicine, University of Chile, CBA No. 274 FMUCH). Liver prolifertion prmeters Restortion of liver mss fter PHx ws determined s percentge of liver mss recovered nd clculted using the rtio of liver weight/body weight of the niml fter PHx. Weight of remnnt liver fter PHx (29.6% of totl weight) ws estimted with 4 nimls euthnized for this purpose. Mitotic figures were counted in HE stined slides, in 3 high power fields (HPF, 4X), nd expressed s the number of mitosis per HPF. Immunohistochemistry studies Stining for Ki-67 nd NF-κB ws performed fter deprffintion, rehydrtion, nd ntigen retrievl in citrte buffer ph 6.1 (1 mm Sodium Citrte,.5% Tween-2) for 3 min t 95 C, endogenous biotin ws blocked with Biotin Blocking System solution (Dko, CA, USA). The nti-ki67 stining ws performed ccording to mnufcturer s instructions (Dko, CA, USA). Positive Ki67 nuclei of heptocytes were counted under light microscope in 1 djcent HPF per slide. This prmeter ws expressed s N of positive cells per HPF. NF-κB p65 Polyclonl ntibody (Thermo Scientific, IL, USA) ws used ccording to the mnufcturer s instruction. A positive control of liver submitted to ischemi reperfusion, with known ctivtion of NF-κB 1 nd negtive control without the primry ntibody were included. Anlysis ws performed

3 Hormonl pretretment fter prtil heptectomy., 213; 12 (6): under light microscope in 1 djcent HPF per slide. Heptocytes re Heptocytes re (size in µm 2 ) ws ssessed with MicrometricsTM softwre in hemtoxylin-eosin stined slides, nd observed under light microscope t higher mgnifiction (4 x) in t lest five djcent fields. Liver injury ssessment Asprtte mino trnsferse (AST) nd lnine mino trnsferse (ALT) ctivities were mesured with commercil kits (Vltek Dignostics, Sntigo, Chile) nd expressed s U/L serum. Liver tissue smples for histology were fixed in formlin, prffin included, cut nd stined with hemtoxiline-eosin (HE). Histologicl evlution ws performed in blind fshion, recording the prmeters liver rchitecture, necrosis, inflmmtion nd stetosis Oxidtive protein dmge Oxidized proteins content ws determined in frozen tissue, treted with 2, 4 dinitrophenylhydrzine to form Schiff bse. Production of the corresponding hydrzone ws mesured spectrophotometriclly between 35 nd 39 nm to determine concentrtion of crbonyls, nd t 28 nm to determine totl protein concentrtion. 11 Vlues were expressed s nmol crbonyls/mg protein. Liver function Serum Albumin quntifiction ws crried out with Rt Albumin ELISA Kit (Abcm, Cmbridge, USA) following the supplier s instructions. Vlues were expressed s mg/dl. TNF-α nd IL-6. RT-PCR studies for inflmmtion induced proteins Liver mrna ws extrcted with kit (EZNA Tissue RNA Kit, Omeg Bio-tek, USA) using supplier s instructions, totl extrcted RNA ws quntified spectrophotometriclly (26/28 nm) nd purity nd integrity confirmed. cdna ws synthesized with Super Script II system (Invitrogen Life Technologies Inc., CA, USA) with hexnucleotides s rndom primers (Rndom Primer). Obtined cdna ws used in chin rection (PCR) using Go- Tq System (Promeg, Mdison, USA), nd specific primers for genes 18S (F 5 -GGA CAT CTA AGG GCA TCA CA-3 ) TNF (tumor necrosis fctor)-α (F 5 -AAA TGG GCT CCC TCT ATC AGT TC-3 ), IL (interleukin)-6 (F 5 -TCC TAC CCC AAC TTC CAA TGC TC-3 ), in Biometr TPersonl thermocycler (Goettingen, Germny). Ech PCR product (25 bp) ws loded in.8% grose gel. Electrophoresis ws performed for 2 min t 8 Volts, visulizing the smples by stining with ethidium bromide. 18 s ws used s control of 35 bp. Bnds were nlyzed by densitometry using the Scion Imge progrm. Quntifiction ws conducted ccording to the reltionship IL-6/18 s, TNF-α/18s s pproprite nd expressed s rbitrry densitometric units (DU). Sttisticl nlysis Sttisticl nlysis ws performed with Grphpd Prism 4 softwre (CA, USA). Vlues shown represent the men ± SD or SEM for the number of seprte experiments indicted; one-wy ANOVA nd the Newmn-Keuls test ssessed the sttisticl significnce of differences between men vlues. P <.5 ws considered significnt. RESULTS Recovery rte of liver mss, estimted s percentge of liver weight to body weight of the niml, ws mesured 16, 24, 72 nd 12 h fter the PHx (Figure 1). In control conditions, liver weight (LW) corresponds to 3.8% of rt body weight (BW), rtio indicted with dotted line. In pnel A, continuous increse in the recovery of liver mss through hours ws observed in ll groups, which reched norml LW/BW rtio fter 12 h post PHX. In pnels B to E the detil of LW/BW for ech time is shown: t 16 hours post PHx (Figure 1B), the group pretreted with MP+T3 showed higher LW/BW rtio (2.135 ±.19%) compred to groups pretreted with T3 nd MP, while t 24 h (Figure 1C) no significnt differences between groups were observed. At 72 h post PHx (Figure 1D), the group pretreted with T3 lone showed significntly decresed LW/BW rtio (2.758 ±.63%) compred to its vehicle nd groups treted with MP lone (3.435 ±.18%) or combined tretment MP+T3 (3.313 ±.113%). At bsl conditions nd 16h fter PHx scrce mitotic figures were observed. Significnt chnges in

4 884 D Espessilles A, et l., 213; 12 (6): A B D % Liver weigh/body weight post PHx % LW/BW 16h post PHx % LW/BW 72h post PHx b b,c,e Hours fter PHx C 4 b E %LW/BW 24h post PHx % LW/BW 12h post PHx T3 b. NOH c. MP d. vmp e. MP + T3 f. vmp + NOH Figure 1. Liver mss recovery fter prtil heptectomy in nimls receiving combined hor- monl pretretment. Progression of liver weight recovery, s liver weight to body weight rtio, fter prtil heptectomy (A), nd detil t 16 (B), 24 (C), 72 (D) nd 12 h (E) fter PHx. Results re shown in percentge of liver weight to body weight rtio (LW/BW). Dotted line indictes norml LW/ BW = 3.8%. Ech br represents the men ± SEM (n = 4). The letters on ech br represent significnt differences between the groups (p <.5, one-fctor ANOVA nd Newmn Keuls test). mitosis counting in groups T3 vs. NOH were observed t 24 (T3, 1.2 ±.51; NOH,.2 ±.82) nd 72h (T3, 1.55 ±.47; NOH,.38 ±.21) post PHx. At 72h T3 mitotic counting ws lso incresed significntly compred with the combined tretment group (MP+T3,.75 ±.13) while t 12h MP+T3 mitotic counting (.3 ±.15) ws higher thn T3 (.14 ±.11) nd MP (.8 ±.1). All results re expressed s men number of mitosis per HPF ± stndrd devition. Figure 2 shows representtive pictures of Ki67 IHC (immunohistochemistry) for the treted groups t 24, 72 nd 12 h post PHx nd the Ki67 number of positive nuclei (indicted by rrows) per HPF, counted in 1 HPF, s men ± SD corresponding to ech group. At 24 h post PHx n incresed number of positive nuclei in ll groups compred to their controls, ws observed; lthough vlues found with MP tretment (Figure 2D) were significntly higher thn T3 (Figure 2A) nd MP+T3 (Figure 2G). At 72 h post PHx, T3 vlues (Figure 2B) (33.2 ± 7.4) were higher thn its control NOH (12.45 ± 4.15 positive nuclei/field) nd MP group (Figure 2E). At 12 h no chnges were observed between tretments or with their respective controls (pnels C, F, I). Cell size, expressed s µm 2, ws nlyzed t 24, 72 nd 12 h post PHx (Figure 3). T3 tretment incresed cell size t ll times compred to its control group nd MP lone. At 24 nd 12 h (Figure 3A nd 3C) the cell size of the T3 treted groups ws lso significntly higher thn the group receiving MP + T3. The combined tretment lso showed n incresed cell size compred to its control nd MP lone t 72 h (Figure 3B) post PHx (p <.5). Activity of serum trnsminses ALT nd AST ws nlyzed 16 nd 24 h fter PHx (Figure 4). T3 significntly decrese ALT levels (66.63 ± U/L), compred with its vehicle (368 ± U/L) t 16 h, but not t 24 h post PHx (pnel A). ALT levels were decrese significntly by the combined pretretment (248 ± 2.65 U/L) compred with its vehicle (623 ± U/L) nd T3 lone (537 ± 16.2) 24h fter

5 Hormonl pretretment fter prtil heptectomy., 213; 12 (6): Hours fter PHx T3 MP MP + T3 Figure 2. Liver Ki67 immunostining fter prtil heptectomy in nimls receiving combined hormonl pretretment. Representtive microphotogrphs of Ki67 positive cells t 24, 72 nd 12 h post PHx. Ech photogrph is representtive of nuclei counting in 1 HPF of n = 3 slides per condition (in prenthesis, men ± SD of nuclei counting, higher mgnifiction of 4x). Cell size μm 2 24h post PHx 1,5 1, 5 A b,c,e Cell size μm 2 72h post PHx 1,5 1, 5 B b,c e f Cell size μm 2 12h post PHx 1,5 1, 5 C b,c,e. T3 b. NOH c. MP d. vmp e. MP + T3 f. vmp + NOH Figure 3. Heptocytes size chnges fter prtil heptectomy in nimls receiving combined hormonl pretretment. Cell size (µm 2 ) ws mesured fter 24 (A), 72 (B) nd 12 h (C) of PHx. Ech br represents men ± SEM (n = 3 slides) of re estimted in 1 HPF (4X). Letters on ech br represent significnt differences between the groups (p <.5, one-fctor ANOVA nd Newmn Keuls test). PHx (pnel B). No significnt chnges were observed in AST levels. Figure 5 shows totl content of oxidized proteins 16 nd 24 h fter PHx. At 16h (pnel A), only the combined pretretment group, MP+T3 (7.56 ±.79 nmol crbonyl/mg protein) vried significntly from its control vmp+noh (13.69 ±,849 nmol crbonyl/mg protein); while t 24 h fter PHx (pnel B)

6 886 D Espessilles A, et l., 213; 12 (6): A C Serum ALT ctivity (U/L) 16h post PHx Serum AST ctivity (U/L) 16h post PHx 1,5 1, 5 1,5 1, 5. T3 b. NOH c. MP d. vmp e. MP + T3 f. vmp + NOH b B D Serum ALT ctivity (U/L) 24h post PHx Serum AST ctivity (U/L) 24h post PHx 1,5 1, 5 1,5 1, 5 e f Figure 4. Serum trnsminses ALT nd AST ctivities fter prtil heptectomy in nimls receiving combined hormonl pretretment. Serum levels of ALT nd AST fter 16 (A, C) nd 24 h (B, D) of heptectomy. Ech br represents men ± SEM (n = 4). Letters on ech br represent significnt differences between the groups (p <.5, one-fctor ANOVA nd Newmn Keuls test). A Liver protein crbonyl content 16h post PHx (nmol/mg protein) f B Liver protein crbonyl content 24h post PHx (nmol/mg protein) b. T3 b. NOH c. MP d. vmp e. MP + T3 f. vmp + NOH Figure 5. Liver oxidized proteins content fter prtil heptectomy in nimls receiving combined hormonl pretretment. Content of crbonylted proteins fter 16 (A) nd 24 (B) h of PHx. Ech br represents men ± SEM (n = 4). Letters on ech br represent significnt differences between the groups (p <.5, one-fctor ANOVA nd Newmn Keuls test). T3 pretretment (2.48 ± 61 nmol crbonyl/mg protein) significntly decrese the vlue of oxidized proteins compred to NOH (1.79 ± 1.23 nmoles crbonyl/mg protein). Liver rquitecture nd presence of necrosis, inflmmtion foci nd stetosis were evluted per group in 3-4 slides, 24 nd 12 h post PHx, nd expressed s men score of 1 HPF, ccording to modified

7 Hormonl pretretment fter prtil heptectomy., 213; 12 (6): Ishk score 12 (Tble 1). 24 h post PHx ll groups hve preserved liver rchitecture, nd presence of mild microvesiculr stetosis. Necrosis foci were more frequently observed in NOH compred with T3, nd were bsent in MP group, while in combined vs. control tretment the presence of necrosis ws similr. Mild to moderte presence of inflmmtory foci ws observed in T3 nd NOH, while the Tble 1. Necrosis, inflmmtion nd stetosis score. The vlues correspond to the verge histologicl nlysis of 3-4 slides 3-4. Reference vlues: = bsent, 1 = mild, 2 = moderte, 3 = severe (modified from Ishk, reference 11). Hours fter PHx Tretment Necrosis Inflmmtion Stetosis 24 T NOH MP 1 vmp MP + T vmp + NOH T3 NOH MP 1 vmp.5 MP + T3 vmp + NOH A B Densitometric Units Liver TNFα (TNFα/18s) 16h post PHx Densitometric Units Liver TNFα (TNFα/18s) 24h post PHx) e d,e C TNF-α 18s TNF-α 18s. T3 b. NOH c. MP d. vmp e. MP + T3 f. vmp + NOH T 3 NOH MP vmp MP+T3 V+V T 3 NOH MP vmp MP+T3 V+V 16h 24h Figure 6. Liver TNF-α expression by RT-PCR fter prtil heptectomy in nimls receiving combined hormonl pretretment. Ech br represents the men ± SEM (n = 4) densitometric units t 16 (A) nd 24 (B) h fter PHx. The letters on ech br represent significnt differences between groups (p <.5, one-fctor ANOVA nd Newmn Keuls test). Photogrphs in (C) show representtive bnds in grose gels.

8 888 D Espessilles A, et l., 213; 12 (6): post PHx (Figure 6, pnels B nd C) the combined tretment decresed TNF-α expression (.23 ±,98 DU) compred to T3 (.8 ±.44 DU) nd MP (.65 ±.24 DU). Expression level of IL-6 s densitometric units of IL-6/18s t 16 nd 24 h post PHX for ech group did not hve significnt differences between pretreted nd control groups or pretreted groups compred between them (dt not shown). NF-κB nucler stining ws negtive in ll groups (Figure 7, pnels A to F), showing not significnt nucler trnsloction t 24 h fter prtil heptectomy in ny of the groups or controls. The presence of low to moderte cytoplsmic stining is lso present in the negtive control, therefore, is not specific of cytoplsmic presence of the nucler fctor. NF-κB nucler stining ws lso performed t 16h with similr results (dt not shown). DISCUSSION Figure 7. Liver NF-κB immunostining fter prtil heptectomy in nimls receiving combined hormonl pretretment. Representtive microphotogrphs of NF-κB stined slides t 24 h post PHx re shown (pnels A to F). Ech photogrph is representtive observtion of 5 HPF of n = 3 slides per condition. Positive (pnel G) nd negtive (pnel H), re included. tretment with MP lone or combined with T3 presented isolted foci of inflmmtory infiltrte. At 12h the pretreted groups (T3, MP nd MP+T3) hve norml liver rchitecture, without necrosis or inflmmtory infiltrte. Persistent, mild microvesiculr stetosis nd slight disorgniztion, with isolted bllooned heptocytes ws observed in MP nd vmp+noh groups. Liver function ws not compromised in ny of the groups fter PHx, s determined by concentrtion of serum lbumin nlyzed t 16 nd 24 h, which vlues were within the norml rnge of 3.8 to 4.8 mg of lbumin per dl serum (dt not shown). TNF-α expression t 16h ws not significntly different in pretreted groups compred with their respective controls (Figure 6, pnels A nd C). At 24 h In this experimentl study, we tested the effect of combined hormonl pretretment composed of methylprednisolone nd triiodothyronine (MP+T3) over helthy liver regenertion cpcity fter prtil heptectomy (PHx). The chnges observed in terms of liver mss recovery showed tht the tretments, either pplied seprtely or combined, my hve n effect in the recovery speed rte, but do not ffect the finl result, since ll groups reched bsl vlues of LW/ BW fter 12h of prtil heptectomy (Figure 1). The sttisticl significnce observed between the combined tretment compred with ech drug lone t 16h my not be of biologicl relevnce, since no differences were observed compred with the combined vehicle (Figure 1B). At 72h the group treted with T3 hd lower LW/BW index, which my suggest dely in the process of recovery (Figure 1D); however, ll groups reched norml vlues fter 12 h of PHx (Figure 1E). This result ws confirmed by Ki67 immunostining, which showed differences in erlier, but no lter times in terms of positive nuclei per field (Figure 2). Mitosis counting showed differences compred to Ki67 results, nd no return to bsl conditions ws observed in ny of the groups t 12h; however, the lower number obtined t this point suggest tht the return to rest conditions my occurred shortly lter. Regrdless the differences in the prolifertion process progression, hormonl therpy MP+T3, either pplied combined or not, does not compromise liver regenertion. The evlution of erlier points my be of interest to determine if

9 Hormonl pretretment fter prtil heptectomy., 213; 12 (6): differentil entrnce in the cell cycle exists or modifictions of the pthwys involved in the priming phse occur. Cell size ws significntly incresed t ll times by T3 pretretment (Figure 3), which is consistent with literture reports, where T3 induces direct hyperplsi nd hypertrophy. 13,14 This fctor should be considered within the liver mss increse chieved by the tretment with T3 lone. Weight recovery (Figure 1), mitosis count, positive Ki67 nuclei (Figure 2) nd heptocytes size (Figure 3) coincide with norml prmeters found in the liver regenertion process in helthy nimls. 15 Hormonl tretment ffects norml regenertion: exogenous supplementtion of T3 cts s primry mitogen. 13,16,17 T3 (t doses of.2 mg/kg) stimultes prolifertion by ccelerting the induction of cyclin D1, 17 nd lso triggers Cdk-2 expression through Küpffer cell-dependent TNF-α secretion, JNK phosphoryltion nd AP-1 ctivtion. 18 Exogenously pplied prior to PHx, T3 shows the sme ptterns of ccelertion of heptic regenertion. 19 The evidence suggests T3 stimultes DNA synthesis by different pthwys tht ultimtely converge with those involved post PHx Administrtion of high dose of MP prior PHx decreses prolifertion of heptocytes nd reduces liver regenertion in dult rts fter PHx; 22 however, the dministrtion of corticoids pre or post surgery my inhibit overproduction of inflmmtory cytokines such s TNF-α, nd reduce surgicl stress. 23,24 Other uthors hve shown tht, in low doses, MP does not chnge liver mss recovery fter PHx, 25 nd my even ct s fctor fcilitting the recovery of the impired liver regenertion fter rdiofrequency bltion. 26 Our results confirm steroids my ct s nti inflmmtory but not nti proliferting drugs t low doses. The comprison of both drugs used, T3 nd MP, in terms of mss recovery fter PHx, hs not been done before, the results found suggest blnce of individul effects with the dosge used, which my hve its explntion in the equilibrium of cytokines production needed for liver prolifertion, nd the differentil effect of the drugs over these prmeters. As for injury prmeters, T3 nd MP+T3 tretments decrese ALT serum levels nd oxidized proteins content erly fter PHx (Figures 4 nd 5). The protective effect of T3 hs been seen in ischemi-reperfusion liver injury, where it diminishes trnsminses nd up to 91% of the totl content of oxidized proteins. 27 MP tretment in neuronl tissue hs shown protective ction, inhibiting lipid peroxidtion; 28 which my occur in our model. The effects observed with both drugs combined do not suggest ddition, synergy or potentition. Histologicl nlysis confirms the previous results (Tble 1): T3, MP nd the combined tretment decrese inflmmtion nd necrosis foci. Microvesiculr stetosis ws evident in ll groups, which is concordnt with the trnsient ccumultion of lipid in heptocytes, chrcteristic of erly regenertion, which my represent n energy source tht supports the growth. 29 Additionlly, no deleterious effects were found in liver function. TNF-α levels were decresed t 24h fter PHx by the combined tretment, while T3 nd MP lone groups exhibited higher levels of the cytokine. This my be relted with the observtion of less inflmmtory foci observed in tissue slides, lthough, it should be interpreted with cution, since TNF-α is lso required for the progression of prolifertion nd ctivtes protective pthwys. 3 This event will require further reserch to elucidte the role of MP+T3-triggered TNF-α in the prolifertive response. The expression levels of IL-6 were not significntly different in pretreted nd not treted nimls, this my be due to n erlier ctivtion of these genes, necessry to trigger the prolifertive cycle, chrcteristiclly found between 6 minutes nd 8 h post PHx. 31 The trnscription fctor NF-κB is essentil for liver prolifertion. Mice with heptocyte-specific disruption of the inhibitory complex subunit NEMO (NF-κB essentil modifier) hve higher mortlity fter 5% heptectomy nd impired liver regenertion. 32 Although NF-κB is relted to liver inflmmtion nd crcinogenesis, its ctivtion, induced by TNF-α, is lso importnt in the priming phse of liver regenertion, up to 12 h fter PHx, which my explin why we hve not observed NF-κB nucler stining t 16 nd 24h fter PHx. 33 At these points, hormonl tretment did not increse NF-κB nucler trnsloction. We hypothesized tht this trnscription fctor dul role in liver prolifertion my be relted to the time frme of ctivtion, the cell in which it is ctivted nd the level of ctivtion, in similr wy described for free rdicls. 34 In different system, gut ischemi-reperfusion, dul role for NF-κB hs lso been described. 35 Additionl studies re needed to test this hypothesis. In conclusion, we hve found preclinicl evidence tht supports the clinicl use of MP+T3 s prt of hormonl resuscittion protocols dministered to multiorgn donors with helthy livers, nd prticu-

10 89 D Espessilles A, et l., 213; 12 (6): lrly when occurrence of smll for size syndrome is suspected. Even when it is rgued tht T3 is not beneficil in this scenrio, 36 the protection ginst injury nd oxidtive stress we report, which might be relted to nti-inflmmtory properties nd dose dependent, is fctor to be considered when evluting the indiction of such protocols. FINANCIAL SUPPORT Supported by Fondecyt Grnts N nd REFERENCES 1. vn der Hoeven JA, Molem G, Ter Horst GJ, Freund RL, Wiersem J, vn Schilfgrde R, Leuvenink HG, et l. Reltionship between durtion of brin deth nd hemodynmic instbility on progressive dysfunction nd incresed immunologic ctivtion of donor kidneys. Kidney Int 23; 64: Novitzky D, Cooper DK, Rosendle JD, Kuffmn HM. Hormonl therpy of the brin-ded orgn donor: experimentl nd clinicl studies. Trnsplnttion 26; 82: Rosendle JD, Kuffmn HM, McBride MA, Chblewski FL, Zroff JG, Grrity ER, Delmonico FL, et l. 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