Effects of hyperbaric oxygen therapy on the liver after injury caused by the hepatic ischemia-reperfusion process 1

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1 6 ORIGINAL ARTICLE ISCHEMIA-REPERFUSION Effects of hyperbaric oxygen therapy on the liver after injury caused by the hepatic ischemia-reperfusion process 1 Marina Rodrigues Garcia da Silveira I, Maria Rita Margarido I, José Carlos Vanni II, Ricardo Nejo Junior III, Orlando de Castro-e-Silva IV DOI: I Undergraduate Medical student of the Faculty of Medicine of Ribeirão Preto, University of São Paulo (FMRP-USP), Ribeirão Preto-SP, Brazil. Data collection and analysis. II Liver Transplantation Unit Ribeirão Preto Medical School - University of São Paulo, Brazil. III Graduate Student of the medicine school at Universidade Santo Amaro, São Paulo - SP, Brazil. Data collection and analysis. IV Chairman and Head of the Department of Surgery and Anatomy of FMRP USP, Ribeirao Preto - SP, Brazil. Conception. Responsible for English language; Responsible for intellectual and scientific content of the study; Critical Review; Responsible for English language. ABSTRACT PURPOSE: To evaluate the effects of hyperbaric oxygen on rats submitted to hepatic ischemia and reperfusion. METHODS: Twenty-three Wistar rats were divided at random into 3 groups: SHAM, rats submitted to surgical and anesthetic stress without induction of hepatic ischemia/reperfurion; I/R, rats submitted to total ischemia of the hepatic pedicle for 25 min followed by 5 min of reperfusion; HBOI/R, rats submitted to 60 min of hyperbaric oxygen therapy at a pressure of 2 absolute atmospheres immediately after the experimental protocol of ischemia/reperfusion. Hepatic function was evaluated by quantitation of serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST), and by mitochondrial function through the determination of states 3 and 4 of mitochondrial respiration, respiratory control ratio (RCR) and mitochondrial swelling. Data were analyzed by the Mann-Whitney test, with the level of significance set at p <0.05. RESULTS: There was a significant difference in state 3 values for the SHAM group vs I/R and I/R vs IRHBO, in state 4 values for the SHAM group vs I/R; and in mitochondrial swelling for the SHAM groups vs I/RHBO, SHAM vs I/R, and IR vs I/RHBO. CONCLUSION: The use of hyperbaric oxygen after I/R improved in a relative manner both the production of energy and the effects on the mitochondrial wall. Key words: Mitochondria. Ischemia. Reperfusion. Hyperbaric oxygen therapy. Liver. Acta Cirúrgica Brasileira - Vol. 29 (supl. 1)

2 Silveira MRG et al. Introduction Hyperbaric oxygen therapy (HBO) is a treatment based on high concentrations of partial oxygen pressure inside a compartment known as hyperbaric chamber 1-5. The oxygen used in the chamber is pure and is present at a pressure of two atmospheres. Among the multiple indications of HBO are osteomyelitis, acute vasculites caused by medications and biological toxins, and gaseous gangrene, in addition to cases of acute anemia in which blood transfusion is not possible 1-5. The ischemia-reperfusion (I/R) process is essential for certain surgical procedures such as partial liver resections and organ transplantation 1-2. Liver transplantation is a condition during which the injury generated by this process is observed, with its intensity and types of prevention and treatment still being controversial subjects 1. It is believed that submission of tissues and organs to a high oxygen concentration after I/R may improve or at least alleviate the effects of ischemia and, mainly, reperfusion 4,5. Tissue hypoxia is considered to generate certain elements that alter the target organ anatomically and functionally and these elements are considered to be exacerbated by tissue reperfusion, aggravating the hepatic injury. Ischemia is related to the triggering of a series of biochemical reactions that ultimately injure the hepatic tissue, while reperfusion is related to the generation of free radicals, among other factors 5-7. The I/R process involves changes ranging from microvascular alterations to the activation of neutrophils, platelets, Kupffer cells and sinusoidal endothelial cells 7-9. Thus, the objective of the present study was to investigate the effect of the use of HBO after an I/R process. Methods proportion,administered at the dose of 100 mg/kg body weight. A median laparotomy was performed, extending from the inferior third of the xiphoid appendix to the pubis in the superoinferior direction, followed by exploration of the abdominal cavity, delicate dissection of the round ligament of the liver and identification of the hepatic pedicle. The hepatic pedicle was fully clamped for 25 min with with a home-made clamp. The animal was then submitted to 5 min of reperfusion and sacrificed by total exsanguination by puncture of the inferior vena cava after exposure of the vein by manipoulation of the abdominal viscera 10. The HBO procedure was based on the simultaneous exposure of three rats to oxygen at an atmospheric pressure of 2 ATA in a hyperbaric chamber (Sechrist, modelo 2500 B) directly pressurized with oxygen. Each session lasted 60 min, divided into 15 min of compression followed by 30 min of exposure to high-pressure oxygen and 15 min of chamber decompression, corresponding to 30 uninterrupted min of HBO at 2 ATA Hepatic function was determined by measuring serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 13 by analysis of the following parameters of mitochondrial function in hepatic tissue: states 3 and 4 of mitochondrial respiration, respiratory control ratio (RCR), and mitochondrial swelling 14. The results of the biochemical tests were analyzed statistically by the nonparametric Mann-Whitney test, with the level of significance set at 5% (p<0.05). The results were analyzed statistically using the Prisma GraphPad 4.0 software (GraphPad Software Inc, CA). Results Analysis of the values presented in Figure 1 revealed that there was a difference in the four parameters investigated according to the process to which the animals were submitted. Twenty-three male Wistar rats weighing 200 to 300 g were used. The animals were divided into three groups: SHAM rats submitted to surgical and anesthetic stress without exposure to HBO or clamping of the hepatic pedicle; I/R rats submitted to 25 min of ischemia followed by 5 min of reperfusion, with no exposure to HBO; I/R-HBO rats submitted to 25 min of ischemia and 5 min of reperfusion, followed by 60 min of HBO at 2 absolute atmosheres (ATA). Duly cleaned but not sterilized surgical material was used for the operation in a standardized manner. The animals were anesthetized with a solution of xylazine hydrochloride (20 mg/ml) and ketamine hydrochloride (50 mg/ml) at a 1: Acta Cirúrgica Brasileira - Vol. 29 (supl. 1) 2014

3 Effects of hyperbaric oxygen therapy on the liver after injury caused by the hepatic ischemia-reperfusion process FIGURE 1 - Hepatic mitochondrial function of the groups studied: SHAM (rats submitted to surgical stress without exposure to hyperbaric oxygen therapy (HBO) or clamping of the hepatic pedicle), IR (rats submitted to 25 min of ischemia followed by 5 min of reperfusion, with no exposure to HBO), and IR+HBO (rats submitted to 25 min of ischemia followed by 5 min of reperfusion and then to 60 min of HBO at 2 absolute atmospheres. State 3 of mitochondrial respiration: SHAM vs IR (p<0.05), SHAM vs IR+HBO (p>0.05) and IR vs IR+HBO (p<0.05). State 4 of mitochondrial respiration: IR vs IR+HBO (p>0.05), SHAM vs IR+HBO (p>0.05) and SHAM vs IR (p<0.05). Respiratory control ratio (RCR): SHAM vs IR (p>0.05), IR vs IR+HBO (p>0.05) and SHAM vs IR+HBO (p>0.05). Mitochondrial swelling: SHAM vs IR (p<0.05), SHAM vs IR+HBO (p<0.05) and IR vs IR+HBO (p<0.05). As shown in Figure 1, it can be seen that the rate of oxygen consumption and the consequent production of energy (State 3) in the form of adenosine triphosphate (ATP) differed significantly between groups (SHAM vs IR and IR vs IR+HBO), being more exacerbated in the SHAM and IR groups compared to the IR and IRHBO groups. The rate of oxygen consumption in the basal state, represented by the graph for state 4, differed significantly between the SHAM and IR groups, since the SHAM and IR+HBO groups and the IR and IR+HBO groups did not show a difference. Regarding mitochondrial swelling, the associations between SHAM and IR, SHAM and IR+HBO, and IR and IR+HBO presented differences, the most marked being between the SHAM and IR groups. The RCR did not differ between groups. Discussion The present study intended somehow to clarify a topic of extreme importance such as hepatic injury due to I/R. This complex that affects the liver in various surgical procedures such as liver transplantation 15 is being investigated in order to find some way of attenuating its adverse effects. In an attempt to contribute to a greater number of possibilities for the attenuation of the undesirable effects of the procedure, we performed the surgical procedure of hepatic I/R followed by submission of the experimental animals to high oxygen pressures in a hyperbaric chamber 10,12,16. Previous clinical studies by our group have shown a beneficial effect of HBO for the treatment of patients with post-liver transplant complications. The experiment involved the analysis of parameters related to mitochondrial respiration such as State 3, State 4 and mitochondrial swellling related to calcium homeostasis and RCR In the present study, the production of energy by the mitochondria was analyzed starting from State 3. Regarding this analysis, we may state that the animals submitted to HBO after hepatic I/R showed an improvement when compared to the rats submitted only to I/R. This demonstrates that the capacity of respiration and, consequently, of energy production was improved in animals submitted to higher oxygen pressure after clamping and unclamping of the hepatic pedicle. In contrast, comparison of the IR group to the SHAM group revealed a reduced capacity of energy production, which was preserved in the HBO+IR group. On this basis, we may conclude that HBO theoretically attenuated the Acta Cirúrgica Brasileira - Vol. 29 (supl. 1)

4 Silveira MRG et al. effects of the period of ischemia due to the promotion of aerobic metabolism by preserving the ATP reserves 3. Regarding State 4 of mitochondrial respiration, there was an expressive difference betweeen the SHAM and IR groups (p<0.05), with a reduced oxygen consumption in the IR group due to interruption of blood flow. Although expected, no difference was observed with HBO. This fact was not observed in other studies, in which the SHAM and IR groups already presented some difference 10-12,16. In those studies there was an increase in the rate of oxygen consumption by the mitochondria compared to the SHAM group in order to maintain the electrochemical proton gradient on the mitochondrial membrane so that the membrane would be able to phosphorylate ADP 16,17. RCR also did not differ significantly between the groups studied. Treatment under hyperoxia did not demonstrate an improvement compared to the group submitted to IR. Regarding this parameter, the SHAM group (p>0.05) was practically similar to the IR group. Regarding the swelling of the mitochondial matrix, considerable differences were detected between the SHAM, IR and IR+HBO groups. This parameter is induced by calcium and phosphate ions which act on this process related to cell permeability. In this respect, it was observed that there was less swelling in the IR and IR+HBO groups than in the SHAM group. However, the IR+HBO group was found to be closer to the SHAM group, leading us to conclude that the wall injury was attenuated by HBO, since the group was comparable to the IR group, whose reduced absorbance induced by calcium and phosphate was lower, showing that the mitochondria of this group were already swollen. On this basis, it is quite likely that the supply of oxygen at high pressure after I/R injury may act as a protector of the mitochondrial membrane. This protection is due to the fact that oxygen acts as an agent that permits the formation of ATP by means of oxidative phosphorylation, as also demonstrated in other studies 2,3,17. This provides the energy needed for the occurrence of ion flow through the mitochondrial membrane, with consequent fluid inflow into the mitochondria. The disequilibrium that occurs in this process may be due to oxidative damage, with further reduction of the utility of the transporters. Conclusion Experiments involving ischemia/reperfusion followed by hyperbaric oxygen revealed a relative improvement of the functional parameters of hepatic mitochondria both regarding energy production and damage to the mitochondrial wall. References 1. Castro-Silva O, Sankarankutty AK, Martinelli AL, Souza FF, Teixeira AC, Feres O, Mente ED, Oliveira GR, Akita R, Muglia V, Elias JJr, Ramalho LN, Zucoloto S. Therapeutic effect of hyperbaric oxygen in hepatic artery thrombosis and functional cholestasis after orthotopic liver transplantation. Transplant Proc. 2006;38(6): Tibbles PM, Edelsberg JS. Hyperbaric oxygen therapy. N Engl J Med. 1996;334(25): Castro-Silva O, Centurion S, Pacheco EG, Brisotti JL, Oliveira AF, Sasso KD. Aspectos básicos da lesão de isquemia e reperfusão e do pré-condicionamento isquêmico. Acta Cir Bras. 2002;17: Thom SR. Hyperbaric oxygen: its mechanisms and efficacy. Plast Reconstr Surg. 2011;127(1): Lima CX, Sanches MD, Rezende Neto JB, Oliveira e Silva RC, Teixeira MM, Souza DG, Santos DC, Melo JR. Hyperbaric oxygen therapy aggravates liver reperfusion injury in rats. Acta Cir Bras. 2008;23: Kanoria S, Glantzounis G, Quaglia A, Dinesh S, Fusai G, Davidson BR, Seifalian AM. Remote preconditioning improves hepatic oxygenation after ischaemia reperfusion injury. Transpl Int. 2012;25(7): Theodoraki K, Tympa A, Karmaniolou I, Tsaroucha A, Arkadopoulos N, Smyrniotis V. Ischemia/reperfusion injury in liver resection: a review of preconditioning methods. Surg Today. 2011;41(5): Tolentino EC, Castro-Silva O, Zucoloto S, Souza ME, Gomes MC, Sankarankutty AK, Oliveira GR, Feres O. Effect of hyperbaric oxygen on liver regeneration in a rat model. Transplant Proc. 2006;38: Yu SY, Chiu JH, Yang SD, Yu HY, Hsieh CC, Chen PJ, Lui WY, Wu CW. Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion injury in rats. J Surg Res. 2005;128: Caldeira DES, Souza MEJ, Gomes MCJ, Picinato MANC, Fina CF, Feres O, Castro-e-Silva O. Effects of hyperbaric oxygen ( HBO ), as pre-conditioning in liver of rats submitted to periodic liver ischemia/ reperfusion. Acta Cir Bras. 2013;28(1): Baldim LB, Nejo R, Souza MEJ, Gomes MCJ, Picinato MANC, Fina CF, Castro-e-Silva O. Effect of hyperbaric oxygen therapy on liver function during intermittent ischemia. Acta Cir Bras. 2013;28: Losada DM, Souza MEJ, Gomes MCJ, Picinato MANC, Fina CF, Feres O, Castro-e-Silva O. Hyperbaric oxygen therapy and ischemia and reperfusion: a valuable association to attenuate ischemic lesion and hepatic reperfusion. Acta Cir Bras. 2013;28(2): Henry RJ, Chiamori N, Golub OJ, Berkman S. Revised spectrophotometric methods for the determination of glutamicoxalacetic transaminase, glutamic-pyruvic transaminase, and lactic dehydrogenase. Am J Clin Pathol. 1960;34: Souza ME, Polizello AC, Uyemura SA, Castro-Silva O, Curti C. Effect of fluoxetine on rat liver mitochondria. Biochem Pharmacol. 1994;48: Castro-e-Silva O, Mente ED, Sankarankutty AK, Souza MEJ, Gomes MCJ, Picinato MANC, Fina CF, Lopes JR. Biochemical liver function after partial hepatic resection with or without partial hepatic vascular exclusion. Acta Cir Bras. 2011;26: Fujita N, Nagatomo F, Murakami S, Kondo H, Ishihara A, Fujino H. Effects of hyperbaric oxygen on metabolic capacity of the skeletal muscle in type 2 diabetic rats with obesity. ScientificWorldJournal. 2012;2012: Ozden TA, Uzun H, Bohloli M, Toklu AS, Paksoy M, Simsek G, Durak H, Issever H, Ipek T. The effects of hyperbaric oxygen treatment on oxidant and antioxidants levels during liver regeneration in rats.tohoku J Exp Med. 2004;203(4): Acta Cirúrgica Brasileira - Vol. 29 (supl. 1) 2014

5 Effects of hyperbaric oxygen therapy on the liver after injury caused by the hepatic ischemia-reperfusion process Correspondence: Orlando de Castro e Silva Jr Departamento de Cirurgia e Anatomia Faculdade de Medicina de Ribeirão Preto, USP Campus Universitário Ribeirão Preto - SP Brasil Tel.: (55 16) orlando@fmrp.usp.br 1 Research performed at Liver Transplantation Unit, Department of Surgery and Anatomy, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (FMRP-USP), Ribeirao Preto-SP, Brazil. Acta Cirúrgica Brasileira - Vol. 29 (supl. 1)

Effect of hyperbaric oxygen therapy on liver function during intermittent ischemia 1

11 ORIGINAL ARTICLE ISCHEMIA-REPERFUSION Effect of hyperbaric oxygen therapy on liver function during intermittent ischemia 1 Efeito da oxigenoterapia hiperbárica sobre a função hepática na isquemia intermitente