Η αναγέννηση, με τον τρόπο που αυτή επιτελείται στο ήπαρ, δηλαδή με πολλαπλασιασμό των ώριμων κυττάρων όλων των κυτταρικών ομάδων του οργάνου, είναι μία μοναδική ιδιότητα. Πιθανώς η ιδιότητα αυτή να είναι γνωστή από αρχαιοτάτων χρόνων, όπως συμβολίζεται στον μύθο του Προμηθέα. Απόλυτα δικαιολογημένο, εκ τούτου, είναι το μεγάλο ερευνητικό ενδιαφέρον απέναντι στη μοναδική αυτή διεργασία. Το συνηθέστερο μοντέλο που χρησιμοποιήθηκε για τη μελέτη της αναγέννησης είναι η χειρουργική ηπατεκτομή σε μικρά τρωκτικά (κατά κύριο λόγο στον επίμυ). Μετά τη διενέργεια της επέμβασης παρατηρείται συγχρονισμένη είσοδος των ηπατοκυττάρων –αρχικά- και των λοιπών κυτταρικών ομάδων -στη συνέχεια- στη φάση G1 του κυτταρικού κύκλου και σε προετοιμασία πολλαπλασιασμού. Στην πρώτη αυτή φάση τα ηπατοκύτταρα γίνονται δεκτικά στη δράση μίας πλειάδας αυξητικών παραγόντων. Αυτή είναι η εναρκτήρια φάση της αναγέννησης (priming phase). Ακολουθεί η φάση πολλαπλασιασμού ή μεταβολική φάση, όπου λόγω των μεγάλων ενεργειακών αναγκών των διαιρούμενων κυττάρων, επισυμβαίνουν χαρακτηριστικά μεταβολικά γεγονότα (παροδική υπογλυκαιμία, συστηματική λιπόλυση και παροδική ηπατοκυτταρική στεάτωση), για να καλύψουν τις ανάγκες αυτές. Στο διάστημα του πολλαπλασιασμού εμφανίζονται τα παρακρινικά και τα αυτοκρινικά σήματα μεταξύ των διαφορετικών κυτταρικών ομάδων του ήπατος. Στα τρωκτικά η φάση αυτή ολοκληρώνεται 4 ημέρες μετά την ηπατεκτομή και ακολουθεί η τρίτη και τελευταία φάση του τερματισμού της αναγέννησης. Τότε συμβαίνει η πολυπαραγοντική ρύθμιση της λήξης του πολλαπλασιασμού. Με εκπληκτική ακρίβεια ρυθμίζεται το βάρος του ήπατος σε συνάρτηση με τη συνολικό βάρος του ζώου, με χρήση και ενός κύματος απόπτωσης, ενώ ακολουθεί αποκατάσταση της φυσιολογικής σύστασης της εξωκυττάριας ουσίας και της ιστολογικής δομής του ηπατικού ιστού. Σε ένα αντικείμενο τόσο διεξοδικά μελετημένο, εντοπίστηκε ένα νέο πεδίο έρευνας που υιοθετήθηκε στην παρούσα διατριβή: ο πιθανός ρυθμιστικός ρόλος των microRNAs στην αναγέννηση του ήπατος. Τα microRNAs είναι μικρά μόρια μη κωδικοποιητικού RNA (μήκους 22 περίπου νουκλεοτιδίων), που ανακαλύφθηκαν σχετικά πρόσφατα. Ωστόσο, με γοργούς ρυθμούς αποκαλύπτεται ο μείζονος σημασίας ρυθμιστικός ρόλος τους στην έκφραση των γονιδίων και άρα στη ρύθμιση πολλαπλών κυτταρικών λειτουργιών. Κατά την έναρξη της παρούσας διατριβής υπήρχαν στοιχεία που ενέπλεκαν τα microRNAs στην αναγέννηση των πτερυγίων του είδους ψαριών zebrafish, τη αναγέννηση των σκωλήκων Planaria spp. και στην επούλωση του τραύματος. Διατυπώθηκε η υπόθεση ότι μπορεί να έχουν ρυθμιστικό ρόλο και στην ηπατική αναγέννηση και μεγάλο μέρος της μελέτης αφιερώθηκε στη διαλεύκανση του ρόλου αυτού. Πρώτο μέλημα των ερευνητών ήταν η βελτιστοποίηση και τυποποίηση της αναισθησιολογικής και εγχειρητικής διεργασίας, που για τον μυ δεν ήταν τόσο διαδεδομένες όσο ήταν για τον επίμυ, λόγω της δυσκολίας που παρουσιάζει η διενέργεια χειρουργικής επέμβασης σε ένα ζώο βάρους 20 γραμμαρίων. Έγιναν πολλαπλές τροποποιήσεις στις παλαιότερες τεχνικές, με αποτέλεσμα την τυποποίηση μίας διαδικασίας που εγγυάται την ταχύτατη διενέργεια της επέμβασης (12-15 λεπτά) με άριστη (95-100%) επιβίωση των πειραματόζωων. Με χρήση της προαναφερθείσας χειρουργικής μεθόδου διενεργήθηκε η πρώτη εγχειρητική πειραματική διαδικασία: Χρησιμοποιήθηκαν 56 πειραματόζωα, τα μισά εκ των οποίων υποβλήθηκαν σε 2/3 μερική ηπατεκτομή και τα υπόλοιπα μισά σε επέμβαση Sham. Λήφθηκαν τα δείγματα ηπατικού ιστού, στον χρόνο 0 και για τα χρονικά σημεία μετά αναγέννηση 1, 3, 6, 12, 24, 36, 48 ωρών, από 4 πειραματόζωα για κάθε χρονικό σημείο. Η πρώτη χρήση των δειγμάτων ιστού από το πρώτο πείραμα έγινε η επιβεβαίωση της συγκρισιμότητας των αποτελεσμάτων του νέου χειρουργικού μοντέλου με αυτά της διεθνούς βιβλιογραφίας. Έγινε ανοσοϊστοχημική χρώση για ανάδειξη της πρωτεΐνης Ki-67 και άρα της χρονικής αλληλουχίας του ρυθμού πολλαπλασιασμού των ηπατοκυττάρων. Αναδείχθηκε, όπως αναμενόταν, η 36η ώρα μετά την ηπατεκτομή ως το χρονικό σημείο που ο μέγιστος αριθμός ηπατοκυττάρων βρίσκεται σε φάση πολλαπλασιασμού στον μυ. Για περαιτέρω επιβεβαίωση του χειρουργικού μοντέλου, στη συνέχεια έγινε ημιποσοτική εκτίμηση της χρονικής εξέλιξης της παροδικής ηπατοκυτταρικής στεάτωσης μετά από χρώση αιματοξυλίνης-ηωσίνης. Από την αξιολόγηση των αποτελεσμάτων προκύπτει ότι η μέγιστη συσσώρευση λίπους ανευρίσκεται, όπως αναμενόταν, στα χρονικά σημεία 12 και 24 ωρών (+++). Η μελέτη, στη συνέχεια, στράφηκε στην κατεύθυνση αξιολόγησης του ρόλου των microRNAs. Για τον σκοπό αυτό ακολούθησε η δεύτερη εγχειρητική πειραματική διαδικασία. Χρησιμοποιήθηκαν 20 πειραματόζωα εκ των οποίων τα μισά υποβλήθηκαν σε 2/3 μερική ηπατεκτομή ενώ τα υπόλοιπα σε επέμβαση Sham. Μετά από αναγέννηση 12 ωρών λήφθηκαν οι ηπατικοί ιστοί για μελέτη του προφίλ έκφρασης των microRNAs. Η επιλογή των 12 ωρών έγινε ως ένα χρονικό σημείο κατά τη φάση έναρξης της αναγέννησης, αλλά όχι στα πολύ αρχικά της στάδια, με γνώμονα την αναζήτηση του τυχόν ρυθμιστικού ρόλου των microRNAs. Το προφίλ έκφρασης των microRNAs μελετήθηκε με τη μέθοδο των μικροσυστοιχιών. Ελέγχθηκαν τα 598 microRNAs που ήταν γνωστά κατά τον καιρό της μελέτης. Τα αποτελέσματα ανέδειξαν ότι εμφανίζεται διαφορική έκφραση σε 8 microRNAs κατά την αναγέννηση. Αναλυτικότερα, τα mmu-miR-21 και mmu-miR-30b εμφάνισαν μεγαλύτερη έκφραση, ενώ τα υπόλοιπα 6 miRNAs (mmu-miR-34c, mmu-miR-144, mmu-miR-207, mmu-miR-451, mmu-miR-582-3p, mmu-miR-290-5p) εμφάνισαν μικρότερη έκφραση κατά την αναγέννηση. Τα δείγματα ιστών του δεύτερου πειράματος χρησιμοποιήθηκαν εκ νέου για επιβεβαίωση των ανωτέρω αποτελεσμάτων με τη μέθοδο RT-qPCR. Η qPCR επιβεβαίωσε το προφίλ έκφρασης των διαφορικά εκφρασμένων miRs, όπως είχαν δείξει τα δεδομένα από τα microarrays. Προέκυψε επίσης ότι το πιο σημαντικά διαφοροποιημένο mmu-miR μεταξύ αυτών που μελετήθηκαν, ήταν το mmu-miR-21. Για να συνδεθούν τα διαφοροποιημένα microRNAs με τις κυτταρικές λειτουργίες στις οποίες εμπλέκονται και πιθανώς ρυθμίζουν, εκτελέστηκε Gene Ontology ανάλυση με τη βοήθεια του TergetScan. Προέκυψε πλειάδα δυνητικών στόχων για τα εν λόγω microRNAs, με σαφή σχέση των γονιδίων-στόχων με τη διεργασία του κυτταρικού πολλαπλασιασμού. Από τα ως τότε αποτελέσματα, τράβηξε την προσοχή η μεγάλη μεταβολή στην έκφραση του mmu-miR-21. Αυτό, σε συνδυασμό με την γνωστή από άλλες μελέτες, εμπλοκή του mmu-miR-21 στο αναπαραγωγικό δυναμικό καρκινικών κυττάρων, αποφασίστηκε να αναζητηθεί η χρονική αλληλουχία έκφρασής του, με χρήση των δειγμάτων ηπατικού ιστού από το πρώτο πείραμα. Αρχικά, χρησιμοποιήθηκε η μέθοδος της RT-qPCR, που ανέδειξε σαφή υπεροχή της έκφρασης του mmu-miR-21 στις 12 ώρες μετά από τη μερική ηπατεκτομή με διατήρηση σχετικά υψηλής συγκέντρωσης ως και τις 24 ώρες. Ακολούθησε επιτυχής επιβεβαίωση του ανωτέρω αποτελέσματος με τη χρήση της μεθόδου του in situ υβριδισμού. Συμπερασματικά, η παρούσα μελέτη πέτυχε να υποδείξει μία πολύ αποτελεσματική μέθοδο 2/3 ηπατεκτομής στον μυ. Το χειρουργικό αυτό μοντέλο αποδείχθηκε ότι έχει πλήρως συγκρίσιμα αποτελέσματα με αυτά της διεθνούς βιβλιογραφίας. Στη συνέχεια επιβεβαιώθηκε η υπόθεση διαφορικής έκφρασης των microRNAs κατά τη διαδικασία της αναγέννησης. Το γεγονός αυτό, υπονοεί ότι ίσως να εμπλέκονται με κάποιο ρυθμιστικό ρόλο στη διαδικασία αυτή. Το mmu-miR-21 αναδεικνύεται ως το μάλλον σημαντικότερο από αυτά. Τα δεδομένα από την παρούσα μελέτη συμπληρώνουν τις έως τώρα γνώσεις για το φαινόμενο της ηπατικής αναγέννησης, αλλά και ανοίγουν δρόμους για νέο προσανατολισμό στην έρευνα, όπως την παραπέρα διαλεύκανση του τρόπου δράσης αυτών των microRNAs που εντοπίστηκαν ή τον τυχόν ρόλο τους και στις φάσεις πολλαπλασιασμού ή τερματισμού της αναγέννησης. / Liver regeneration is a unique ability, because of the way it proceeds, i.e. the proliferation of all categories of all mature liver cell types. It is highly possible that this ability is known to human kind since the ancient times, as pictured in Prometheus’ myth. The great scientific interest towards deciphering this complex process is, of course, highly justified. The most common model for the study of the process of regeneration is the surgical model of the 2/3 partial hepatectomy (PHx) in small rodents, predominantly the rat. 2/3 partial hepatectomy leads to a highly synchronized hepatocyte cell-cycle entry and progres¬sion. The first phase, known as the ‘priming phase’, occurs in the first hours after PH and poises the hepatocytes to enter the G1 phase and to become receptive to growth factors. The second phase corresponds to an increased metabolic demand imposed on the remnant liver. During this phase, among other metabolic changes, transient hypoglycemia is suggested to induce systemic lipolysis followed by a lipid droplets accumulation in the hepa¬tocytes. During this phase, am major role is played by the autocrine intercellular network. In rodents, this phase is completed in 4 days post-PHx and is followed by the termination phase. Ending the regenerative process is an equally complex, multiparameter process. The weight of the liver is regulated proportionally to the animal’s body weight with remarkable accuracy, sometimes employing an apoptotic wave. The termination phase of the regenerative process ends with normal hepatic histological structure restoration and matrix remodeling. Liver regeneration is a phenomenon that has been thoroughly studied in the past. Nevertheless, a point of emerging research interest has been adopted in the present study: the possible regulatory role of microRNAs in liver regeneration. MicroRNAs are small non-coding RNA molecules (approx. 22 nts long), which have been discovered quite recently but through research they are quickly emerging as cornerstone regulatory means in a large number of cellular functions. In the beginning of this study, data existed implicating microRNAs in the regeneration of zebrafish fins, regeneration in planarian worms and wound healing. The hypothesis that they may have a role in liver regeneration was made and a large part of this study is concerned with investigating the existence of such a role. The researchers began with revising and standardizing the method for anesthesia and surgical procedure, which, at the time (2007), were not satisfactory enough in the case of mice (as opposed to the widely used rats), possibly because of the difficulty of operating on a 20 gram animal. Many alterations were made upon the previous techniques. As a result, a procedure was standardized, as described herein, that guarantees a fast procedure (12-15 minutes) accompanied by excellent animal survival (95-100%). Using the above described technique, the first surgical experiment in this study was conducted: 56 animals (wild-type mice) were used, half of which were subjected to 2/3 PHx and the other half were sham operated. Liver samples were collected at time 0 and at several time points during regeneration (1, 3, 6, 12, 24, 36, 48 hours), with a number of 4 animals per time point. These samples were used in order to confirm the comparability of the new surgical technique to bibliography models. An immunohistochemical dye for the protein Ki-67 was performed, thus revealing the number of hepatic cells undergoing proliferation at each time point. The 36th hour post-PHx emerged as the point of climax of the proliferative process in the mouse, as expected by previous studies. For further confirmation, the same samples were used to produce simple histological H-E slides, in order to evaluate the evolvement of lipid droplet accumulation in hepatic cells associated with liver regeneration. A semi-quantitative evaluation was conducted, that revealed that maximum lipid accumulation occurs at the time points of 12 and 24 hours (+++) in mouse, again as expected by previous studies. Then the study proceeded with investigating the potential role of microRNAs in liver regeneration. For this, a second surgical experiment was conducted: This time 20 animals (wild-type mice) were used, half of which were subjected to 2/3 PHx and the other half were sham operated. After regenerating for 12 hours, liver samples were harvested from all animals. The choice of the 12-hour interval was made as a time point at the beginning phase of liver regeneration, but not at the very early beginning, with a view to reveal the possible regulatory role of microRNAs at the first stage of the regenerative process. MicroRNA profiling was conducted using specific microarrays, examining the presence of the 598 microRNAs known at the time of this procedure. The results pointed out 8 differentially expressed microRNAs during regeneration: 2 that were up-regulated (-miR-21 and mmu-miR-30b) and 6 that were down-regulated (mmu-miR-34c, mmu-miR-144, mmu-miR-207, mmu-miR-451, mmu-miR-582-3p, mmu-miR-290-5p). Tissue samples from the second experiment were used again in order to confirm the aforementioned results utilizing the RT-qPCR method. This indeed confirmed the microarrays’ results and highlighted mmu-miR-21 as the most differentially expressed miR, indicating a possibly major regulatory role in liver regeneration. In order to link these differentially expressed microRNAs to their cellular and molecular functions, Gene Ontology Analysis was conducted, using TargetScan. Many putative gene-targets for each microRNA emerged, many of which are involved in the process of cellular proliferation. Following the emergence of the major differentiation of mmu-miR-21 within the results of qPCR evaluation and with previous research linking it with cancer cell proliferation regulation, it was decided to further assess the time kinetics of the expression of mmu-miR-21, utilizing tissue samples from the first experiment. Through an RT-qPCR evaluation, it was shown that up-regulation of mmu-miR-21 reaches its zenith at 12 hours post-PHx and remains quite highly expressed until 24 hours. This was further confirmed by in situ hybridization. In conclusion, we were able to standardize a very successful version of the 2/3 hepatectomy procedure adapted for mice. Using this model, the hypothesis of the altered expression of microRNAs during liver regeneration is confirmed, setting suspicion about some kind of regulatory role. Mmu-miR-21 emerges as the most differentially expressed one and possibly having the most important role. The data from the present study supplement preexisting knowledge on the phenomenon of liver regeneration, but also show the way for future research in further clarifying the paths leading to microRNAs’ regulatory role or investigating their potential role in the phases of proliferation or termination of liver regeneration.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O fÃgado possui uma notÃvel capacidade de regeneraÃÃo apÃs trauma tecidual, incluindo hepatectomia parcial. EspÃcies reativas de oxigÃnio e peroxidaÃÃo lipÃdica tÃm sido incluÃdas nos mecanismos de proliferaÃÃo e crescimento celulares. DimetilsulfÃxido (DMSO) e ternatina (TRT), conhecidos varredores de radicais livres, e os Ãcidos graxos poliinsaturados (PUFA) w-3 e w-6 foram estudados em um modelo experimental para avaliar suas influÃncias na regeneraÃÃo hepÃtica e no estresse oxidativo. Cento e oito ratos Wistar machos foram aleatoriamente distribuÃdos em seis grupos de dezoito animais. Grupo 01 (G1) foi o grupo controle: os ratos foram somente submetidos à laparotomia (sem hepatectomia parcial) no tempo T0. Todos os outros grupos, alÃm de hepatectomia parcial à Higgins-Anderson (HP) no tempo T0, se submeteram, diariamente por duas semanas, à infusÃo intraperitoneal (i.p.) de uma dada droga: G2 recebeu soro fisiolÃgico 0,9% (salina) 0,1mL/kg, em G3 foi infundido PUFA w-3 0,1g/kg, em G4 foi TRT 1,0mg/kg, G5 DMSO 3,3mg/kg e G6 PUFA w-6 0,1g/kg. Em cada grupo, nos tempos 36h(T1), 168h(T2) e 336h(T3) pÃs-HP, um subgrupo de seis ratos foi escolhido ao acaso para hepatectomia complementar (em G1 foi realizada hepatectomia total), quando sangue e os lobos residuais posteriores do fÃgado foram obtidos para exames e estudos. Todas as intervenÃÃes cirÃrgicas foram realizadas sob anestesia inalatÃria com Ãter dietÃlico. SubstÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) foram mensurados no sangue e no fÃgado. ConcentraÃÃes sangÃÃneas de glicose, bilirrubina total e transaminase glutÃmico-pirÃvica (TGP, transaminase de alanina) foram tambÃm avaliadas como indicadores de dano hepÃtico e de homeostase orgÃnica dependente do fÃgado. Os resultados obtidos foram primeiro submetidos ao teste de Kruskal-Wallis para verificar a normalidade aproximada das distribuiÃÃes e entÃo analisados: a evoluÃÃo ponderal por anÃlise de regressÃo e teste t de Student e os demais parÃmetros por mÃdia + E.P.M. e o teste comparativo de Dunnett: p<0,05 foi aceito como estatisticamente significativo. TRT, DMSO e PUFA w-3 inibiram a regeneraÃÃo hepÃtica. PUFA w-6, ao contrÃrio, nÃo apresentou efeito inibitÃrio. PeroxidaÃÃo lipÃdica aumentou tanto no fÃgado como no sangue com o aporte de w-6.. DMSO, TRT e PUFA w-3 induziram reduÃÃo nas concentraÃÃes de GSH hepÃtico 07 dias pÃs-HP. Os resultados do presente estudo dÃo suporte aos achados de que o estresse oxidativo desempenha um papel importante no processo de regeneraÃÃo hepÃtica pÃs-hepatectomia parcial em ratos. / The liver exhibits a remarkable regenerative capacity after tissue damage, including partial hepatectomy. Reactive oxygen species and lipid peroxidation have been implicated as control mechanisms of cellular growth and proliferation. Dimethylsulfoxide (DMSO) and ternatin (TRT), known free radical scanvengers, and the w-3 and w-6 polyunsaturated fatty acids (PUFA) were evaluated in an experimental model to assess their influence on rat liver regeneration and oxidative stress. One hundred and eight male Wistar rats were randomly assigned to six groups which contained 18 animals each. Group 01 (G1) was the control group: rats were just submitted to laparotomy (without partial hepatectomy) at the time T0. All the others groups, besides Higgins-Anderson partial hepatectomy (HP) at time T0, received daily for fourteen days, by intraperitoneal (i.p.) route, one of the following exogenous drug: G2 got NaCl 0.9% (saline) 0.1mL/kg,, G3 receveid w-3 PUFA 0.1g/kg, G4 TRT 1.0mg/kg, G5 DMSO 3.3mg/kg, and G6 w-6 PUFA 0.1g/kg. In each group, at the time 36h(T1), 168h(T2) and 336h(T3) post-HP, a subgroup of six rats was chosen in a randomized way to complementary hepatectomy (in G1 a total hepatectomy was performed), when blood and the liver residual posterior lobes were taken to studies and exams. All surgical procedures were performed under inhalatory ether anesthesia. Thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) were measured in plasma and in liver tissue. Blood concentrations of glucose, total bilirubin, and serum glutamic pyruvic transaminase (SGPT,alanine transaminase) were also evaluated as indicators of hepatic damage and organic homeostasis liver-dependent. Data were first submitted to Kruskal-Wallis test to verify the approximate normality of the distributions and then they were analyzed: liver weight evolution via analysis of regression and t test of Student and all others parameters by mean + S.E.M. and comparative test of Dunnett: p<0.05 was accepted as statistically significant. TRT, DMSO and PUFA w-3 inhibited the liver regeneration. PUFA w-6, on the contrary, did not show inhibitory effect. Lipid peroxidation got higher in blood and in liver after the administration of w-6. DMSO, TRT, and PUFA w-3 induced a reduction on hepatic GSH concentration 7 days post-HP. The results of the present study reinforce the hypothesis that oxidative stress plays an important role on rat liver regeneration phenomenon after partial hepatectomy.

A perda do parênquima hepático, induzida por tratamento agudo, cirúrgico ou químico, desencadeia um processo regenerativo até que a massa hepática seja completamente restaurada. A regeneração hepática, após a hepatectomia parcial de 70%, é um dos modelos mais adequados de regeneração de células, órgãos e tecidos mais estudados. No fígado, ainda que sejam atribuídas propriedades regenerativas, muitas das lesões são tão prejudiciais que este mecanismo de reparação é insuficiente, tornando o transplante a única opção. As células derivadas do broto hepático de ratos apresentam uma boa alternativa para tratamento de doenças hepáticas devido ao seu alto índice proliferativo e da expressão de marcadores de pluripotência, sendo sua aplicabilidade viável em modelos experimentais. O objetivo deste trabalho foi analisar as diferentes vias de administração das células derivadas do broto hepático de ratos com 12,5 dias de gestação visando a melhor regeneração do órgão. Foram realizados experimentos de hepatectomia parcial de 70% (N=50 animais), PET Scan MSFX PRO In-Vivo RX e fluorescência, índice hepatossomático, análise de marcadores solúveis (GH, AFP, CEA, IGF-1), análises hematológicas, microscopia de luz (coloração HE, Tricômio de Masson), análise de marcadores por citometria de fluxo (CD90, STRO-1, Nanog, Oct3/4, Ki-67, Caspase 3) e ciclo celular por citometria de fluxo. Nossos dados demonstraram que as células do broto hepático administradas na via endotraqueal apresentaram melhor equilíbrio entre proliferação e morte celular, com maior expressão dos marcadores de pluripotência, melhor organização celular e regeneração tecidual, em contraste com outras vias, incluindo endovenosa, intraperitoneal e oroenteral. Isto a torna mais segura e de maior viabilidade na regeneração celular em relação às demais vias, sendo mais eficiente nos modelos de hepatectomia parcial / The restoration of liver parenchyma after partial hepatectomy or chemical treatments represents appropriate models to study regeneration mechanisms. The most appropriate model for liver regeneration is partial hepatectomy of 70%, however, organ repair properties are insufficient, suggesting the transplantation the best alternative to treat liver diseases. Cells derived from liver buds of rats show a high proliferative index and the expression of pluripotency markers; thus their significance for regeneration purposes can be tested experimentally. We here investigated different routes to administer cells derived from rat live buds of 12.5 days of gestation to adult individuals (N=50 animals) suffering from partial hepatectomy (70%). Applied methods included PET Scan MSFX PRO In-Vivo RX, fluorescence hepatossomatic index, analysis of soluble markers (GH, AFP, CEA, IGF-1), hematological analysis, light microscopy (staining HE, and Masson trichrome) as well as flow cytometry for cell cycle analysis and CD90, STRO-1, Nanog, OCT3/4, Ki-67, Caspase 3 expression. Our data showed that administration via the tracheal route resulted as favorite in regard to the balance between proliferation and cell deaths, of pluripotency marker expression, cellular organization and tissue regeneration, in contrast to other routes including: intravenous, intraperitoneal and oroenteral. Consequently, the tracheal route showed safer and more efficient treatment to enhance cell regeneration after partial hepatectomy

Ce travail doctoral est centré sur la régénération du foie en conditions normales et au cours de la stéatose hépatique. Ces dernières décennies, de nombreux travaux ont utilisé des modèles d’invalidations géniques afin d’identifier les acteurs important au cours de la régénération hépatique. Dans ce contexte, il avait été observé que des animaux dont le signal de l’hormone de croissance était inhibé présentait un défaut majeur de prolifération hépatocytaire après hépatectomie. Dans un premier temps, notre laboratoire s’est donc intéressé à comprendre comment l’hormone de croissance contrôle la régénération hépatique au niveau moléculaire. Pour cela, nous avons pratiqué des hépatectomies sur des animaux dépourvus en récepteur de l’hormone de croissance (GHrKO). Nous avons ainsi montré que l’hormone de croissance jouait un rôle majeur au cours de la régénération hépatique en contrôlant l’expression d’EGFR ainsi que l’activation de Erk1/2. Dans un second temps, je me suis intéressée à une situation pathologique associée à une dérégulation de la voie de l’hormone de croissance : la stéatose hépatique. De façon intéressante, de nombreux modèles murins de stéatose hépatique présentent également une inhibition importante de la prolifération après hépatectomie partielle. Chez l’Homme, cette maladie (NAFLD pour Non alcoholic fatty liver disease) représente un facteur de risque lors de transplantations hépatiques et de résections majeures du foie. Grâce à l’analyse quantifiée de plusieurs paramètres issue de biopsies de patients obèses, nous avons montré l’existence d’une forte corrélation entre stéatose hépatique et diminution de l’expression de l’EGFR sur l’Homme. Nous avons également pratiqué des hépatectomies sur deux modèles de stéatose, l’un génétique (ob/ob) l’autre induit par un régime déficient en méthionine et choline (MCD). Les cinétiques de régénération post hépatectomie nous ont permis de confirmer un défaut de régénération hépatique chez les souris ob/ob et MCD. D’autre part, l’étude de ces modèles de stéatose nous a amenés à valider la dérégulation de la voie de l’hormone de croissance et la diminution transcriptionnelle de l’EGFR avant et après hépatectomie partielle. En parallèle, nous avons souligné l’implication de la voie inhibitrice de prolifération TGF-β, dans l’altération de la prolifération hépatocytaire des animaux ob/ob. En effet, de nombreux acteurs de cette voie sont surexprimés après l’hépatectomie partielle, participant certainement au défaut de régénération plus drastique observé sur ce modèle. Pour finir, nous avons également montré que l’injection sur une courte période d’hormone de croissance sur les animaux ob/ob restaure la prolifération hépatocytaire post hépatectomie. Ce sauvetage phénotypique est associé à une réexpression transcriptionnelle et protéique de l’EGFR. A terme, ces travaux nous amènent à proposer que la dérégulation de l’axe hormone de croissance/EGFR représente un mécanisme général associé à la stéatose hépatique et responsable du défaut de régénération du foie lié à cette maladie. / This doctoral work focused on liver regeneration in physiological conditions and during steatosis. These last decades, several studies used gene invalidation models to identify important actors during the liver regeneration. In this context, it had been observed that animals displaying a defect of growth hormone pathway had a drastic defect of liver regeneration after partial hepatectomy. Initially, we started this work by focusing on understanding how growth hormone controls liver regeneration at the molecular level. To do so, we performed partial hepatectomies on animals deleted for the growth hormone receptor gene (GHrKO). These results showed that growth hormone plays a central role in the control of liver regeneration through the expression of EGFR and the activation of Erk1/2. Secondly, we focused our attention on a pathological situation showing a defect of growth hormone signaling : hepatic steatosis. Interestingly, many mice models of hepatic steatosis also present a drastic inhibition of hepatocytes proliferation after partial hepatectomy. In Humans, non-alcoholic fatty liver disease (NAFLD) represents an important risk factor regarding liver transplantations and resections. Through quantified analysis of several parameters from obese patient biopsies, we showed the existence of a strong correlation between hepatic steatosis and decrease in EGFR expression on humans. We also performed partial hepatectomies on two models of hepatic steatosis, one being genetic (ob/ob) and the other one being induced by a methionine choline deficient diet (MCD). Kinetics of regeneration post hepatectomy led us to confirm the defect of liver regeneration in on ob/ob and MCD mice. Moreover, the study of these steatotic models allowed us to corroborate the downregulation of the growth hormone signaling and the transcriptional decrease of EGFR expression. We also underlight the importance of TGF-β, a signaling pathway inhibiting proliferation, in the liver regeneration defect observed in ob/ob mice. Indeed, many members of this pathway have been found to be upregulated after partial hepatectomy, possibly being involved in the drastic regeneration defect observed in ob/ob mice. To finish, we also showed that growth hormone injections on a small period of time in ob/ob mice were capable of rescuing hepatocyte proliferation post hepatectomy. This phenotypic rescue was associated with a reexpression of EGFR at the transcription and protein level. This work led us to propose that the defect of the growth hormone/EGFR pathway represents a general mechanism associated with hepatic steatosis and is responsible for the liver regeneration defect linked to this disease.

Liver resection causes marked perfusion alterations in the liver remnant both on the organ scale (vascular anatomy) and on the microscale (sinusoidal blood flow on tissue level). These changes in perfusion affect hepatic functions via direct alterations in blood supply and drainage, followed by indirect changes of biomechanical tissue properties and cellular function. Changes in blood flow impose compression, tension and shear forces on the liver tissue. These forces are perceived by mechanosensors on parenchymal and non-parenchymal cells of the liver and regulate cell-cell and cell-matrix interactions as well as cellular signaling and metabolism. These interactions are key players in tissue growth and remodeling, a prerequisite to restore tissue function after PHx. Their dysregulation is associated with metabolic impairment of the liver eventually leading to liver failure, a serious post-hepatectomy complication with high morbidity and mortality. Though certain links are known, the overall functional change after liver surgery is not understood due to complex feedback loops, non-linearities, spatial heterogeneities and different time-scales of events. Computational modeling is a unique approach to gain a better understanding of complex biomedical systems. This approach allows (i) integration of heterogeneous data and knowledge on multiple scales into a consistent view of how perfusion is related to hepatic function; (ii) testing and generating hypotheses based on predictive models, which must be validated experimentally and clinically. In the long term, computational modeling will (iii) support surgical planning by predicting surgery-induced perfusion perturbations and their functional (metabolic) consequences; and thereby (iv) allow minimizing surgical risks for the individual patient. Here, we review the alterations of hepatic perfusion, biomechanical properties and function associated with hepatectomy. Specifically, we provide an overview over the clinical problem, preoperative diagnostics, functional imaging approaches, experimental approaches in animal models, mechanoperception in the liver and impact on cellular metabolism, omics approaches with a focus on transcriptomics, data integration and uncertainty analysis, and computational modeling on multiple scales. Finally, we provide a perspective on how multi-scale computational models, which couple perfusion changes to hepatic function, could become part of clinical workflows to predict and optimize patient outcome after complex liver surgery.

INTRODUÇÃO: Transplantes parciais de fígado em crianças têm sido realizados com maior freqüência, enfatizando a importância do estudo da regeneração hepática, bem como dos efeitos de drogas imunossupressoras sobre a mesma. A regeneração do parênquima é o resultado do balanço entre multiplicação celular e apoptose, esta última definida como morte celular programada. Neste processo, estão envolvidas expressões de genes pró-apoptóticos (Bak e Bax) e anti-apoptóticos (Bcl-XL e Bcl-2). Dentre as proteínas relacionadas à proliferação hepatocitária, destaca-se a interleucina-6 (IL-6). Embora o modelo de ressecção de 70% da massa hepática de ratos adultos seja amplamente utilizado para estudos de regeneração, não há trabalhos com animais em crescimento. MÉTODOS: Na presente pesquisa, foi padronizado o modelo de hepatectomia parcial em ratos recém-nascidos e em recém-desmamados: realizou-se ligadura com fio de algodão do pedículo dos lobos esquerdo lateral, esquerdo medial e direito medial, seguida da ressecção do parênquima desses lobos. Os fígados remanescentes foram imediatamente pesados e comparados com os pesos dos fígados de animais controles. Para caracterização dos modelos de regeneração, 40 ratos recém-nascidos e 30 recém-desmamados foram submetidos à hepatectomia descrita e mortos nos dias subseqüentes (1, 2, 3, 4 e 7), e o fígado residual submetido a análises de peso e histologia convencional. A seguir, 36 animais (18 recém-nascidos e 18 recém-desmamados) foram divididos nos seguintes grupos: controle, cirurgia simulada e hepatectomia. Um dia após, utilizando métodos moleculares (técnica do RT-PCR), estudou-se a expressão do gene da IL-6, dos genes pró-apoptóticos e anti-apoptóticos nos fígados desses animais e, por meio de métodos imunoistoquímicos, analisou-se a presença de antígenos relacionados à proliferação celular (PCNA) e apoptose (TUNEL) em lâminas preparadas pela técnica do \"tissue microarray\". Em outros 36 ratos (18 recém-nascidos e 18 recém-desmamados), foram administradas drogas imunossupressoras (metilprednisolona, ciclosporina A ou tacrolimus, separadamente) no ato da hepatectomia, e o parênquima hepático remanescente submetido às mesmas análises moleculares e imunoistoquímicas, um dia após. RESULTADOS: A ressecção do parênquima hepático correspondeu a 70% da massa total do fígado. A mortalidade relacionada à hepatectomia nos animais recém-nascidos e recém-desmamados foi 30% e 0% respectivamente. Na análise histológica observou-se maior quantidade de mitoses em hepatócitos no terceiro dia nos recém-nascidos e no segundo dia nos recém-desmamados, com normalização da arquitetura do parênquima até o 7º dia e recuperação total do peso de ambos. No animal recém-nascido, notou-se intensa esteatose associada ao processo regenerativo. A hepatectomia provocou aumento na expressão do gene da IL-6 no fígado residual e diminuição da expressão dos genes pró-apoptóticos em ambos os modelos, além de aumento do anti-apoptótico Bcl-2 nos animais recém-desmamados. O estudo realizado sobre o efeito das drogas imunossupressoras mostrou resultados diferentes daqueles descritos em animais adultos, não havendo alteração no número de células em proliferação (PCNA positivas) ou apoptose (TUNEL positivas). As drogas não tiveram efeito sobre a expressão do gene da IL-6. Metilprednisolona e tacrolimus ocasionaram aumento da expressão do gene anti-apoptótico Bcl-2 em ambos os modelos; metilprednisolona e ciclosporina provocaram aumento na expressão do gene pró-apoptótico Bak nos ratos recém-nascidos. CONCLUSÕES: os modelos de regeneração hepática em ratos recém-nascidos e recém-desmamados foram exeqüíveis e adequados para a pesquisa; a hepatectomia promoveu estímulo da proliferação de hepatócitos com inibição da apoptose; as drogas imunossupressoras utilizadas não exerceram efeito sobre a proliferação de hepatócitos porém provocaram aumento da expressão de genes relacionados a apoptose. / INTRODUCTION: Partial liver transplantation has been performed in children with increasing frequency, and this emphasizes the importance of the studies of hepatic regeneration and the effects of immunosuppressive drugs on this phenomenon. Liver regeneration is controlled by the balance between cell proliferation and apoptosis (defined as a programmed cell death that results from the expression of pro-apoptotic genes - Bax and Bak - and anti-apoptotic genes - Bcl-2 and Bcl-XL). Among proteins related to hepatocyte proliferation, interleukin-6 is an important one. Although the adult rat model of 70% hepatectomy has been widely utilized for studies of liver regeneration, there are no studies using growing animals. METHODS: In the current paper, two experimental models were created utilizing newborn and weaning rats: using a cotton thread, the vascular hilum and the hepatic vein were ligated and the left lateral, left medial and right medial lobes were resected. The remaining liver was immediately harvested and weighted to be compared to control livers. The animals were sacrificed on days 1, 2, 3, 4, and 7 after the operation, the remnant livers were weighted and harvested for histological examinations. Then, 36 animals (18 newborn and 18 weaning animals) were divided into the following groups: control, sham and hepatectomy. One day after, the expressions of IL-6 gene, pro-apoptotic and anti-apoptotic genes were studied in the remnant livers. Immunohistochemical stainings for cell antigens related to cell proliferation (PCNA) and apoptosis (TUNEL) were also performed utilizing tissue microarray sections. In another group of 36 animals (18 newborn and 18 weaning animals), immunosuppressive drugs were administered just after the hepatectomy (methylprednisolone, cyclosporine A or tacrolimus separately), and the remnant liver submitted to the same molecular and immunohistochemical studies. RESULTS: The resected liver corresponded to 70% of the total liver weight. The mortality rates after hepatectomy were 30% and 0% for newborn and weaning rats, respectively. The histological examinations showed a great number of mitoses of hepatocytes on the third day in newborns and on the second day in weaning rats, and normalization of histological aspects by 7 days after hepatectomy and weight recuperation. In the newborn group liver regeneration was related to an intense steatosis. Hepatectomy promoted an increase in the expression of IL-6 gene of the remnant liver, a decreased expression of pro-apoptotic genes in both models, and an increased expression of anti-apoptotic Bcl-2 gene in weaning rats. The study of the effects of immunosuppressants showed different results from those described in adult animals, with no alterations in the number of cells in proliferation (PCNA positive) and apoptosis (TUNEL positive). Drugs had no effect in expression of IL-6 gene. Methylprednisolone and tacrolimus promoted an increased expression of anti-apoptotic gene Bcl-2. In addition, methylprednisolone and cyclosporine promoted an increase in the expression of the pro-apoptotic gene Bak in newborn rats. CONCLUSIONS: The experimental models were feasible and adequate for the current investigations; hepatectomy stimulated hepatocyte proliferation and inhibited hepatic cells apoptosis; the utilized immunosuppressant drugs did not affect hepatocyte proliferation although an increased expression of apoptosis-related genes was verified.

INTRODUÇÃO: Transplantes parciais de fígado em crianças têm sido realizados com maior freqüência, enfatizando a importância do estudo da regeneração hepática, bem como dos efeitos de drogas imunossupressoras sobre a mesma. A regeneração do parênquima é o resultado do balanço entre multiplicação celular e apoptose, esta última definida como morte celular programada. Neste processo, estão envolvidas expressões de genes pró-apoptóticos (Bak e Bax) e anti-apoptóticos (Bcl-XL e Bcl-2). Dentre as proteínas relacionadas à proliferação hepatocitária, destaca-se a interleucina-6 (IL-6). Embora o modelo de ressecção de 70% da massa hepática de ratos adultos seja amplamente utilizado para estudos de regeneração, não há trabalhos com animais em crescimento. MÉTODOS: Na presente pesquisa, foi padronizado o modelo de hepatectomia parcial em ratos recém-nascidos e em recém-desmamados: realizou-se ligadura com fio de algodão do pedículo dos lobos esquerdo lateral, esquerdo medial e direito medial, seguida da ressecção do parênquima desses lobos. Os fígados remanescentes foram imediatamente pesados e comparados com os pesos dos fígados de animais controles. Para caracterização dos modelos de regeneração, 40 ratos recém-nascidos e 30 recém-desmamados foram submetidos à hepatectomia descrita e mortos nos dias subseqüentes (1, 2, 3, 4 e 7), e o fígado residual submetido a análises de peso e histologia convencional. A seguir, 36 animais (18 recém-nascidos e 18 recém-desmamados) foram divididos nos seguintes grupos: controle, cirurgia simulada e hepatectomia. Um dia após, utilizando métodos moleculares (técnica do RT-PCR), estudou-se a expressão do gene da IL-6, dos genes pró-apoptóticos e anti-apoptóticos nos fígados desses animais e, por meio de métodos imunoistoquímicos, analisou-se a presença de antígenos relacionados à proliferação celular (PCNA) e apoptose (TUNEL) em lâminas preparadas pela técnica do \"tissue microarray\". Em outros 36 ratos (18 recém-nascidos e 18 recém-desmamados), foram administradas drogas imunossupressoras (metilprednisolona, ciclosporina A ou tacrolimus, separadamente) no ato da hepatectomia, e o parênquima hepático remanescente submetido às mesmas análises moleculares e imunoistoquímicas, um dia após. RESULTADOS: A ressecção do parênquima hepático correspondeu a 70% da massa total do fígado. A mortalidade relacionada à hepatectomia nos animais recém-nascidos e recém-desmamados foi 30% e 0% respectivamente. Na análise histológica observou-se maior quantidade de mitoses em hepatócitos no terceiro dia nos recém-nascidos e no segundo dia nos recém-desmamados, com normalização da arquitetura do parênquima até o 7º dia e recuperação total do peso de ambos. No animal recém-nascido, notou-se intensa esteatose associada ao processo regenerativo. A hepatectomia provocou aumento na expressão do gene da IL-6 no fígado residual e diminuição da expressão dos genes pró-apoptóticos em ambos os modelos, além de aumento do anti-apoptótico Bcl-2 nos animais recém-desmamados. O estudo realizado sobre o efeito das drogas imunossupressoras mostrou resultados diferentes daqueles descritos em animais adultos, não havendo alteração no número de células em proliferação (PCNA positivas) ou apoptose (TUNEL positivas). As drogas não tiveram efeito sobre a expressão do gene da IL-6. Metilprednisolona e tacrolimus ocasionaram aumento da expressão do gene anti-apoptótico Bcl-2 em ambos os modelos; metilprednisolona e ciclosporina provocaram aumento na expressão do gene pró-apoptótico Bak nos ratos recém-nascidos. CONCLUSÕES: os modelos de regeneração hepática em ratos recém-nascidos e recém-desmamados foram exeqüíveis e adequados para a pesquisa; a hepatectomia promoveu estímulo da proliferação de hepatócitos com inibição da apoptose; as drogas imunossupressoras utilizadas não exerceram efeito sobre a proliferação de hepatócitos porém provocaram aumento da expressão de genes relacionados a apoptose. / INTRODUCTION: Partial liver transplantation has been performed in children with increasing frequency, and this emphasizes the importance of the studies of hepatic regeneration and the effects of immunosuppressive drugs on this phenomenon. Liver regeneration is controlled by the balance between cell proliferation and apoptosis (defined as a programmed cell death that results from the expression of pro-apoptotic genes - Bax and Bak - and anti-apoptotic genes - Bcl-2 and Bcl-XL). Among proteins related to hepatocyte proliferation, interleukin-6 is an important one. Although the adult rat model of 70% hepatectomy has been widely utilized for studies of liver regeneration, there are no studies using growing animals. METHODS: In the current paper, two experimental models were created utilizing newborn and weaning rats: using a cotton thread, the vascular hilum and the hepatic vein were ligated and the left lateral, left medial and right medial lobes were resected. The remaining liver was immediately harvested and weighted to be compared to control livers. The animals were sacrificed on days 1, 2, 3, 4, and 7 after the operation, the remnant livers were weighted and harvested for histological examinations. Then, 36 animals (18 newborn and 18 weaning animals) were divided into the following groups: control, sham and hepatectomy. One day after, the expressions of IL-6 gene, pro-apoptotic and anti-apoptotic genes were studied in the remnant livers. Immunohistochemical stainings for cell antigens related to cell proliferation (PCNA) and apoptosis (TUNEL) were also performed utilizing tissue microarray sections. In another group of 36 animals (18 newborn and 18 weaning animals), immunosuppressive drugs were administered just after the hepatectomy (methylprednisolone, cyclosporine A or tacrolimus separately), and the remnant liver submitted to the same molecular and immunohistochemical studies. RESULTS: The resected liver corresponded to 70% of the total liver weight. The mortality rates after hepatectomy were 30% and 0% for newborn and weaning rats, respectively. The histological examinations showed a great number of mitoses of hepatocytes on the third day in newborns and on the second day in weaning rats, and normalization of histological aspects by 7 days after hepatectomy and weight recuperation. In the newborn group liver regeneration was related to an intense steatosis. Hepatectomy promoted an increase in the expression of IL-6 gene of the remnant liver, a decreased expression of pro-apoptotic genes in both models, and an increased expression of anti-apoptotic Bcl-2 gene in weaning rats. The study of the effects of immunosuppressants showed different results from those described in adult animals, with no alterations in the number of cells in proliferation (PCNA positive) and apoptosis (TUNEL positive). Drugs had no effect in expression of IL-6 gene. Methylprednisolone and tacrolimus promoted an increased expression of anti-apoptotic gene Bcl-2. In addition, methylprednisolone and cyclosporine promoted an increase in the expression of the pro-apoptotic gene Bak in newborn rats. CONCLUSIONS: The experimental models were feasible and adequate for the current investigations; hepatectomy stimulated hepatocyte proliferation and inhibited hepatic cells apoptosis; the utilized immunosuppressant drugs did not affect hepatocyte proliferation although an increased expression of apoptosis-related genes was verified.

L’enzyme méthionine synthase (MTR) catalyse la reméthylation de l’homocystéine en méthionine, le précurseur du donneur universel de groupe méthyle S-Adenosylmethionine (SAM), impliqué dans des mécanismes de régulations épigénétiques. Des polymorphismes de MTR sont associés à des défauts métaboliques et des défauts de développement embryonnaire. Afin d’étudier les conséquences d’une déficience en MTR, nous avons généré des modèles murins d’invalidation conditionnelle du gène MTR de manière constitutive ou inductible dans le foie et dans le cerveau. L’invalidation constitutive ou inductible ciblée dans le foie pendant l’embryogenèse n’est pas viable, suggérant un rôle limitant de la méthionine synthase sur le développement précoce et l’organogenèse en lien probable avec les conséquences sur la prolifération cellulaire. Dans les périodes post-natales, nous avons utilisé le modèle inductible complété par une hépatectomie pour étudier les altérations de la régénération hépatique liée aux effets sur le stress cellulaire ainsi que l’expression et l’activation des cyclines. Le KO dans le cerveau induit principalement une perte des fonctions de mémorisation de l’apprentissage hippocampo-dépendant. Au total, nos résultats illustrent les effets différents de l’invalidation de MTR en fonction de l’organe considéré. Le foie est un organe très plastique avec une capacité de régénération très importante. Les effets sur les étapes de l’organogénèse et sur l’inhibition de la régénération confirment l’hypothèse du rôle majeur et limitant de la méthionine synthase dans la régulation du cycle cellulaire. Le modèle d’invalidation au niveau du cerveau confirme le rôle très important de la voie de reméthylation de l’homocystéine catalysée par la méthionine synthase, rôle qui a déjà été illustré par d’autres travaux sur les rats carencés en donneur de méthyle et sur la souris transgénique KO cd320 / The enzyme methionine synthase (MTR) catalyzes the remethylation of homocysteine to methionine, the precursor of the methyl donor S-universal Adenosylmethionine (SAM), involved in epigenetic regulation mechanisms. We generated mouse models with conditional invalidation of the mtr gene in a constitutive or inducible manner to delete the gene expression specifically in the liver and brain. Constitutive invalidation during embryonic life is not sustainable when targeted to the liver, suggesting a limiting role of methionine synthase in early organogenesis and probably on cell proliferation. We performed hepatectomy to study regeneration-related effects on the cellular stress and found dramatic effects on cell proliferation through altered expression and activation of cyclins. The constitutive model in brain highlighted the behavioral anomalies related to a loss of learning and memory. This suggested major effects in the hippocampus. Overall, our findings highlighted the specific effects of the invalidation of methionine synthase in both organs. The liver is a plastic member with a very high regenerative capacity. The effects on organogenesis and inhibition of regeneration confirm the hypothesis for a major role of methionine synthase in cell cycle regulation. The invalidation model in the brain confirms the important role of the remethylation pathway catalysed by methionine synthase, a role which has been shown by other studies in rats deprived in methyl donors and in cd320 KO transgenic mice

INTRODUÇÃO: O transplante hepático se consolidou como terapêutica para crianças com doença hepática em estágio terminal. A principal indicação nessa faixa etária é a atresia das vias biliares, doença colestática que leva à cirrose hepática na maioria dos casos nos primeiros anos de vida mesmo nas crianças submetidas a portoenterostomia. Essas crianças evoluem entre outras complicações com desnutrição grave e necessitam do transplante precocemente. A relação ideal do peso do enxerto hepático e o peso do receptor varia de 1 a 3%. No entanto, quando o transplante é realizado em crianças em idade inferior a 2 anos, essa relação na maioria das vezes é maior do que 5%. Essa situação é conhecida como large-for-size, em que a perfusão sanguínea do enxerto pode ser insuficiente, o que leva a disfunção do órgão. Os mecanismos de lesão neste cenário ainda não estão bem esclarecidos. A lesão de isquemia-reperfusão (IR) é apontada como um dos fatores para o mal funcionamento do enxerto bem como de outras complicações. Nessa lesão estão envolvidos elementos da cascata inflamatória culminando em morte celular por apoptose bem como mecanismos de regeneração celular. Um fenômeno conhecido que atenua a lesão de IR é o pré-condicionamento isquêmico (PCI). Nenhum trabalho experimental foi realizado com o objetivo de avaliar a lesão hepática na situação large-for-size e o efeito do PCI nessa situação. MÉTODOS: Para a padronização do modelo foram realizados 8 transplantes, tendo sido testados os pesos dos animais, relação entre o peso do enxerto hepático e peso do receptor e técnica cirúrgica. Na fase experimental, foram realizados 21 transplantes e os animais foram divididos em 3 grupos. No primeiro grupo (controle) o transplante era realizado com doadores e receptores com pesos similares. Para caracterização da situação large-for-size, no segundo grupo os doadores tinham aproximadamente o dobro do peso dos receptores (LFS). No terceiro grupo a distribuição de peso foi semelhante a do segundo, porém era realizado o PCI no doador (LFS+PCI). Os procedimentos foram realizados sob anestesia geral e técnica asséptica e os animais foram mantidos vivos por 3 horas após a reperfusão. Foram obtidas amostras de sangue em 3 períodos (logo após abertura da cavidade abdominal, 1 e 3 horas após reperfusão) e fragmentos de fígado 1 e 3 horas após reperfusão. As amostras de sangue foram submetidas a gasometria arterial, dosagem de sódio, potássio e enzimas hepatocelulares. Os fragmentos de tecido hepático foram submetidos a análise histológica pela coloração da hematoxilina-eosina (HE) e a análise de quantificação da expressão dos genes pró-apoptótico (Bax), anti-apoptótico (Bcl- XL), da óxido nítrico-sintase endotelial (eNOS) e da interleucina-6 (IL-6). RESULTADOS: A mortalidade observada foi de 28%. A relação peso do enxerto e peso do paciente foi de 2,9% no grupo controle, 6,3% no grupo LFS e 6,4% no grupo LFS+PCI. A natremia 1 hora após a reperfusão foi mais elevada no grupo controle do que nos demais, já com relação ao potássio, a dosagem foi mais baixa nesse grupo no mesmo intervalo. A dosagem da aspartato aminotransferase (AST) foi mais elevada nos grupos LFS e LFS+PCI do que no controle. A análise histológica não Resumo demonstrou diferença entre os grupos. A expressão do gene Bax foi mais elevada no grupo LFS do que nos demais, enquanto que a do gene eNOS foi mais acentuada no grupo LFS+PCI do que nos demais nos dois intervalos estudados. Após 3 horas de reperfusão a expressão do gene IL-6 foi maior no grupo LFS+PCI. CONCLUSÕES: O modelo de transplante hepático large-for-size em suínos foi exequível e adequado para a pesquisa. A lesão IR foi mais acentuada no grupo LFS do que no grupo controle e apesar de não ter alterado a elevação de enzimas hepatocelulares, o PCI teve papel de aumentar a expressão dos genes IL-6 e eNOS e diminuir a do gene Bax / INTRODUCTION: Liver transplantation has been established as therapy for children with end-stage liver disease. The main indication is biliary atresia, cholestatic disease that leads to cirrhosis in most cases even in children undergoing portoenterostomy. These children develop other complications as severely malnutrition and need early transplantation. The ideal graft to body weight ratio (GBWR) for transplantation varies from 1 to 3%. However, when transplantation is performed in children younger than 2 years, this ratio in most cases is higher than 5%. This situation, known as large-for-size, may be related to insufficient graft perfusion and liver disfunction. The mechanisms of injury involved in this scenario are not well established. Ischemia-reperfusion injury (IRI) has been related as a factor for poor graft function and other complications. Inflammatory cascade culminating in cell death by apoptosis and cellular mechanisms of regeneration are the elements involved in IRI. Ischemic preconditioning (IPC) is a phenomenon that attenuates IR injury. There are no experimental studies conducted to evaluate the hepatic injury in large-for-size situation and the effect of IPC in this situation. METHODS: 8 procedures were required for standardize the model. Weight of the animals, GBWR and surgical technique were evaluated. In the experimental phase, 21 transplantations were performed. The animals were divided in three groups. In the first group (control), liver transplantation was performed with donors and recipients with similar weights. To characterize large-for-size situation, donors of the second group had approximately twice the weight receptor (LFS). In the third group the weight distribution was similar to the second but IPC was performed in donor (LFS+IPC). Procedures were performed under general anesthesia and aseptic technique and the animals were kept alive for 3 hours after reperfusion. Blood samples were obtained at three times (immediately after opening the abdominal cavity, 1 and 3 hours after reperfusion) and fragments of liver 1 and 3 hours after reperfusion. Blood samples were analyzed for arterial blood gases, sodium, potassium and hepatocellular enzymes. The fragments of liver tissues were subjected to histological analysis by hematoxylin-eosin staining (HE). Molecular biology involved quantification of the expression of Bax, Bcl-XL, endothelial nitric oxide synthase (eNOS) and interleukin- 6 (IL-6) genes. RESULTS: The mortality rate was 28%. The GBWR was 2.9% in the control group, 6.3% in the LFS group and 6.4% in the LFS+ICP group. The natremia 1 hour after reperfusion was higher in the control group and potassium dosage was lower in this group in the same period. The dosage of aspartate aminotransferase (AST) was higher in LFS and LFS+IPC groups than control. Histological analysis showed no difference between groups. The Bax gene expression was higher in the LFS, while the eNOS gene was more expressed in LFS + PCI group. After 3 hours of reperfusion the expression of IL-6 gene was higher in the PCI + LFS. CONCLUSIONS: The model of large-for-size liver transplantation in swine was feasible and appropriate for the research. IR injury was more pronounced in the Summary group LFS than in the control group and despite not having changed the elevation of hepatocellular enzymes, the PCI was responsible for increasing the expression of IL- 6 and eNOS and decreasing the Bax gene expression

A Glutamina (Gln) melhora a regeneração hepática em ratos nutridos. Para avaliar o efeito molecular da Gln endovenosa no remanescente hepático, 52 ratos foram classificados em: 1. Nutridos com hepatectomia parcial (HP) e Gln (N-Gln=10) ou prolina (N-Pro=10); 2. Desnutridos com HP: (D-Gln=10) ou (D-Pro=10); 3. Nutridos e Desnutridos sem HP (N-Controle=6) e (D-Controle=6). Após 96 horas da HP os resultados foram: DNA = (D-Gln, D-Pro e D-Controle < N-Gln, N-Pro e N-controle), (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln); RNA= (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln e N-Pro). Não houve diferença nos genes transcritos (GT) para HGF, TGF-a e timidina kinase. Concluímos que: A desnutrição e HP reduzem DNA e RNA; A Gln não altera os GT estudados em ratos desnutridos 96 horas após HP / Glutamine dipeptide (Gln) improve hepatic regeneration of nourished rats. To evaluate molecular effect of Gln on liver remnant 52 rats was classified into: 1. Nourished with partial hepatectomy (PH) and Gln (N-Gln=10) or proline (N-Pro=10); 2. Malnourished (MN) with PH: (MN-Gln=10) or (MN-Pro=10); 3. Nourished and Malnourished rat without PH (N-Control=6) and (MN-Control=6). Results: DNA = (MN-Gln, MN-Pro and MN-Control < N-Gln, N-Pro and N-control), (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln); RNA= (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln and N-Pro). There was no difference on transcript genes (TG) for HGF, TGF-a and thymidine kinase. Conclusions: Malnutrition and PH decrease hepatic DNA and RNA; Gln does not modify TG studied 96 hours after PH in MN rats