Original Article / Liver
Hepatobiliary & Pancreatic Diseases International
Pentoxifylline enhances the protective effects of hypertonic saline solution on liver ischemia reperfusion injury through inhibition of oxidative stress Vinicius Rocha-Santos, Estela RR Figueira, Joel A Rocha-Filho, Ana MM Coelho, Rafael Soraes Pinheiro, Telesforo Bacchella, Marcel CC Machado and Luiz AC D'Albuquerque São Paulo, Brazil
BACKGROUND: Liver ischemia reperfusion (IR) injury triggers a systemic inflammatory response and is the main cause of organ dysfunction and adverse postoperative outcomes after liver surgery. Pentoxifylline (PTX) and hypertonic saline solution (HTS) have been identified to have beneficial effects against IR injury. This study aimed to investigate if the addition of PTX to HTS is superior to HTS alone for the prevention of liver IR injury.
AST, IL-6, mitochondrial dysfunction and pulmonary myeloperoxidase 4 hours after reperfusion. Compared with HTS only, HPTX significantly decreased hepatic oxidative stress 4 hours after reperfusion and pulmonary permeability 4 and 12 hours after reperfusion. CONCLUSION: This study showed that PTX added the beneficial effects of HTS on liver IR injury through decreases of hepatic oxidative stress and pulmonary permeability.
METHODS: Male Wistar rats were allocated into three groups. (Hepatobiliary Pancreat Dis Int 2015;14:194-200) Control rats underwent 60 minutes of partial liver ischemia, HTS rats were treated with 0.4 mL/kg of intravenous 7.5% KEY WORDS: pentoxifylline; NaCl 15 minutes before reperfusion, and HPTX group were hypertonic saline solution; treated with 7.5% NaCl plus 25 mg/kg of PTX 15 minutes behepatic oxidative stress; fore reperfusion. Samples were collected after reperfusion for ischemia reperfusion injury; determination of ALT, AST, TNF-α, IL-6, IL-10, mitochondrial pulmonary permeability respiration, lipid peroxidation, pulmonary permeability and myeloperoxidase. RESULTS: HPTX significantly decreased TNF-α 30 minutes after reperfusion. HPTX and HTS significantly decreased ALT,
Author Affiliations: Department of Gastroenterology, Laboratory of Medical Investigations LIM37 Discipline of Liver and Gastrointestinal Transplantation (Rocha-Santos V, Figueira ERR, Coelho AMM, Pinheiro RS, Bacchella T, Machado MCC and D'Albuquerque LAC) and Discipline of Anesthesiology (Rocha-Filho JA), Hospital das Clinicas, University of São Paulo School of Medicine, São Paulo, SP, Brazil Corresponding Author: Joel A Rocha-Filho, MD, Discipline of Anesthesiology, University of São Paulo School of Medicine, Rua Manoel da Nóbrega, 489 apt 21, 04001-083, São Paulo, SP, Brazil (Tel: +55-11-2661-3323; Email:
[email protected]) © 2015, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: 10.1016/S1499-3872(15)60348-4 Published online March 10, 2015.
Introduction
D
espite improvements in organ storage, liver ischemia reperfusion (IR) injury remains an important issue and is a major risk factor for postoperative liver dysfunction after liver surgery and transplantation.[1, 2] The degree of liver injury has a direct association with the inflammatory cascade triggered during the ischemic phase. Interruption of oxygen delivery leads to a reduction in oxidative phosphorylation and cellular ATP storage.[3] In the early phase of reperfusion, reactive oxygen species (ROSs) are formed because of oxidation of hypoxanthine by xanthine oxidase. ROSs ultimately result in lipid peroxidation.[4, 5] It is well established that lipid peroxidation of cellular membranes plays a major role in hepatic oxidative stress.[6, 7] In the later phase of reperfusion, further activation of Kupffer cells and consequent release of inflammatory molecules
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Hypertonic saline and pentoxifylline decrease oxidative stress
promote and amplify hepatocyte injury.[5] Furthermore, local inflammatory disorders are associated with distant organ damage which worsens patient outcomes.[8-10] Lung injury that causes pulmonary edema, intraalveolar hemorrhage, and neutrophil accumulation is also known to occur after liver IR.[11] Therefore, several strategies have been proposed to avoid this complex adverse event. Experimental models demonstrated decreased IR injury when treatments including the use of hypertonic saline solution (HTS) or pentoxifylline (PTX) are used before revascularization. Studies[12-14] showed that small-volume resuscitation with HTS infusion improved hemodynamic parameters, microcirculation disturbances, liver edema, and pulmonary permeability. PTX is a methylxanthine phosphodiesterase inhibitor that prevents vasodilation of the microcirculation, including that of the liver.[15] It has been shown that PTX increases tissue oxygenation and intestinal blood flow while decreasing TNF and microvascular leukocyte accumulation, thereby reducing the inflammatory response.[16-19] The present study aimed to investigate if the addition of PTX to HTS is superior to the use of HTS alone in of the treatment of liver IR injury.
liver volume was performed to avoid splanchnic congestion.[20, 21] Abdominal wall was closed to prevent dehydration during the ischemic period. Intravenous drugs were given via the dorsal penile vein. After 60 minutes of ischemia, the clamp was removed and liver reperfusion was started. Blood and tissue samples were collected at 30 minutes, 4 and 12 hours after the reperfusion. The blood was collected through cardiac puncture, and the animals were euthanized by exsanguination under anesthesia.
Study groups and experimental protocol Animals submitted to liver IR were allocated into three groups (Fig. 1). In the control group (n=24), animals were subjected to 60-minute ischemia. In the HTS group (n=24), animals were subjected to 60-minute ischemia and then received 0.4 mL/kg of intravenous 7.5% NaCl 15 minutes before reperfusion. And in the HPTX group (n=24), animals were subjected to 60-minute ischemia and received 0.4 mL/kg of intravenous 7.5% NaCl plus 25 mg/kg of PTX 15 minutes before the reperfusion. Additionally, 14 animals from a sham-operated group underwent the surgical procedure without pedicled clamping or treatment. The total number of animals including those underwent sham operation was 86.
Liver aminotransferases Serum aspartate aminotransferase (AST) and alanine Animals aminotransferase (ALT) were used as indicators of liver Male Wistar rats were used according to the recommeninjury. AST and ALT activities were assayed 4 hours after dations of the National Council Guide (1996) for the reperfusion by the optimized ultraviolet method (COBAS Care and Use of Laboratory Animals. The rats weighing MIRA, Roche, Rotkrenz, Switzerland) and optimized ac230-270 g were housed in the LIM37 from University cordingly to International Federation of Clinical Chemof São Paulo School of Medicine. They were placed at a istry. The results were expressed in units per liter (U/L). room temperature of 22 ℃ in a 12-hour light/dark cycle, while receiving food and water ad libitum. The experimental protocol was approved by the Ethics Committee of our institution.
Methods
Anesthesia and surgical procedures Intraperitoneal anesthesia was induced in all animals with ketamine 30 mg/kg (Ketalar, Cristália, SP, Brazil) and xylazine 30 mg/kg (Rompum, Bayer, SP, Brazil), followed by orotracheal intubation. Mechanic ventilation was instituted with a tidal volume of 0.08 mL/g body weight, a respiratory rate of 60/min and 21% FiO2 (Small Animal Ventilator Model 683, Harvard Apparatus, Holliston, MA, USA). Body temperature ranging from 35-37 ℃ was monitored with a rectal digital thermometer (YSI Precision 4000A Thermometer, USA). A midline laparotomy was performed and the pedicles of the left lateral and median hepatic lobes were occluded with a 2.5 mm microvascular clamp. Partial ischemia of 70% of the total
Fig. 1. Study design. Control group: animals subjected to 1 hour of liver ischemia and 4 hours of reperfusion. HTS group: animals were treated with 0.4 mL/kg of intravenous 7.5% NaCl 15 minutes before reperfusion. HPTX group: animals were treated with 7.5% NaCl plus 25 mg/kg of PTX 15 minutes before reperfusion. Shamoperated group: animals subjected to surgical manipulation without liver ischemia.
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Serum TNF-α and interleukins TNF-α was quantified 30 minutes after reperfusion using specific ELISA kits (Biosource International Cytoscreen, Camarillo, CA, USA). IL-6 and IL-10 were quantified 4 hours after reperfusion using specific ELISA kits (Biosource International Cytoscreen).
mL/min, using a syringe pump (975 Harvard Apparatus, Holliston, MA, USA), and after that the lung was weighed. One small piece of tissue was removed and dried at 60 ℃ for calculation of total dry weight. The perfused lung (4 μL/mg of tissue) was immersed in formamide (Merck, Darmstadt, Germany) for 24 hours at room temperature. The Evans blue concentration extracted in formamide was quantified spectrophotoLipid peroxidation metrically at 620 nm (ELX808 Ultra Microplate Reader, Lipid peroxidation was used as an indirect mea- Thomas Scientific, USA).[25] surement of oxidative damage induced by ROS. Lipid peroxidation was determined 4 hours after reperfusion Lung myeloperoxidase assay in ischemic and non-ischemic liver samples by the thioThe levels of myeloperoxidase activity were determined barbiturate reaction measuring the formation of maloin lung samples 4 hours after reperfusion. Samples of ndialdehyde (MDA) spectrophotometrically. Samples of 300 mg lung tissue were homogenized for 60 seconds 100 mg liver tissue were homogenized (Polytron PT-2100 homogenizer, Kinematica AG, Luzern, Switzerland) for with Polytron PT-2100 homogenizer in 1 mL of sodium phosphate buffer at pH 6.2. The samples were sonicated 60 seconds in 1 mL of 1.15% KCl buffer, and centrifuged and centrifuged (3000×g, 30 minutes) at 4 ℃. Ten (Sorvall RC-5C Plus centrifuge, Thermo Fischer Scientifmicroliters of supernatant was added to 500 μL of sodium ic Inc., Waltham, MA, USA) at 14 000×g for 20 minutes phosphate buffer (pH 6.2), 700 μL of 0.25% PBS/BSA, at 4 ℃. Two hundred µL of supernatant was added to 1.5 100 μL of o-dianisidine, and 100 μL of 0.05% H2O2. MymL of 0.8% thiobarbituric acid, 200 µL of 8.1% sodium eloperoxidase activity was analyzed by measuring the dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), and change in absorbance at 490 nm caused by the metabo600 µL of distilled water. The mixture was heated for 45 lism of H2O2 in the presence of o-dianisidina.[12, 26] The minutes at 90 ℃. After cooling, the flocculent precipitate results were expressed as optical density (OD) at 490 nm. was removed by centrifugation at 10 000×g for 10 minutes. Absorbance was measured at 532 nm (Ultra Microplate Liver histology Reader ELX 808 from Bio-Tek Instruments, Winooski, Samples of liver ischemic lobes were collected 4 VT, USA) using MDA bis (dimethyl acetal) as external [22] hours after reperfusion and fixed in 10% buffered forstandard. malin for standard hematoxylin and eosin (HE) staining. A single blinded pathologist performed histologic evaluPolarographic study of liver mitochondria ation. Histologic injury was evaluated according to the The mitochondrial oxygen consumption was evaluat- scoring system proposed by Quireze et al.[27] ed 4 hours after reperfusion with a closed reaction vessel (Gilson Medical Electronics, Middleton, WI, USA) fitted Statistical analysis with the Clark oxygen electrode (Yellow Springs InstruData were expressed as mean±standard deviation (SD). ments, Yellow Springs, OH, USA) at 28 ℃ as described Statistical analysis was performed using the R Statistical [23] previously. The respiratory control ratio calculated as Software for Windows, version 2.12. ANOVA was used the rate of oxygen consumption in state 3/oxygen conto evaluate differences in the control, HTS and HPTX sumption in state 4. The adenosine diphosphate/oxygen groups. When both normality and homogeneity assumpconsumption ratio (ADP/O) was calculated as moles of tions of variances were fulfilled, ANOVA was applied. ATP formed per moles of oxygen consumed by mito- If only homogeneity was satisfied, the Kruskal-Wallis chondria.[24] test was applied. After that, multiple comparisons were
Analysis of lung permeability The lung microvascular permeability was quantified 4 and 12 hours after reperfusion by the Evans blue extravasation technique. Evans blue (Sigma Chemical Co.) dissolved in saline was injected via the dorsal penile vein at 20 mg/kg, 15 minutes before euthanasia. Blood samples were collected, the lungs were perfused via the pulmonary artery with 30 to 50 mL of 0.9% NaCl at 10
performed using the parametric Tukey's test or the nonparametric Tukey's test.[28] In cases that both normality and homogeneity were not satisfied, the non-parametric Tukey's test was used for two-by-two comparisons to identify differences between the groups. Student's t test or the Mann-Whitney test was used to evaluate differences between the control and Sham-operated groups. Differences were considered statistically significant when P value was
