- Email: [email protected]
The Effect of Octreotide on Hepatic Ischemia-Reperfusion Injury in a Rabbit Model
The Effect of Octreotide on Hepatic Ischemia-Reperfusion Injury in a Rabbit Model J. Yang, H. Sun, P. Takacs, Y. Zhang, J. Liu, Y. Chang, and K.A. Candiotti ABSTRACT Background. Hepatic ischemic-reperfusion injury (HIRI) is a major cause of morbidity and mortality following liver surgery. Octreotide (Oct) has been reported to improve hepatocellular energy metabolism in a rat HIRI model. This study was designed to evaluate whether Oct could protect the liver of rabbits against ischemic-reperfusion (I/R) injury. Methods. Twenty-four adult New Zealand rabbits were randomly divided into a sham operated group (Control), an ischemia/reperfusion group (I/R), and an ischemia/ reperfusion þ Oct pretreatment group (I/R þ Oct). The hemodynamic (mean arterial pressure [MAP] and heart rate [HR]) changes, liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]) release, inflammatory cytokines (tumor necrosis factor [TNF]a and interleukin [IL]-1b) levels, and endotoxin (ETX) levels were measured during I/R. Results. Compared with the Control group, the MAP decreased and HR increased in I/R and I/R þ Oct groups at ischemia 15 minutes (P < .05) but were less in the I/R þ Oct group relative to the I/R group (P < .05). ALT, AST, LDH, IL-1b, and ETX levels were increased in the I/R and I/R þ Oct groups at ischemia 30 minutes (P < .05), however, the increase was lower in the I/R þ Oct group relative to the I/R group (P < .05). Bcl-2 expression in the I/R þ Oct group was higher compared with other groups (P < .05) and Bax expression in the I/R group was reduced compared with other groups (P < .05). Hepatocellular damage in the I/R þ Oct group appeared to be less than in the I/R group by microscopy. Conclusions. Oct pretreatment attenuated hemodynamic changes and decreased liver enzyme changes induced by HIRI in a rabbit model. The protection mechanisms of Oct may be related to reduced ETX levels, down-regulation of the inflammatory cytokines TNFa and IL-1b, and inhibition of hepatocellular apoptosis, as well as the modulation of the mitochondrion-mediated Bcl-2/Bax apoptosis pathway. Based on our study it appears that Oct may be useful in decreasing liver injury after liver surgery and/or transplantation and may serve as a promising agent against HIRI.
D
URING liver surgery surgeons may perform temporary inflow and/or outflow occlusion by vascular clamping to reduce intraoperative blood loss. This technique can lead to hepatic ischemic-reperfusion injury (HIRI) and may sometimes result in multiple system organ failure and/or a systemic inflammatory response syndrome after surgery.1 Due to the risks arising from these complications, therapeutic strategies to reduce HIRI have become a priority. The most promising protective strategy against ischemicreperfusion (I/R) injury explored in the last few years appears to be preconditioning, which presumably functions
From the Department of Anesthesiology of Affiliated Tumor Hospital of Xiang-Ya Medical College of Central South University, Changsha, China (J.Y., H.S., J.L.); Department of Anesthesiology of University of Miami Miller School of Medicine, Miami, FL, USA (P.T., Y.Z., K.A.C.); and Second Xiangya Hospital of Xiangya Medical School, Central South University, Changsha, China (Y.C.). Address reprint requests to Jinfeng Yang, MD, PhD, Department of Anesthesiology of Affiliated Tumor Hospital of Xiang-Ya Medical College of Central South University, 283# Tongzipo Rd., Yuelu Dist., Changsha, China 410013. E-mail: 315977705@qq. com
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.02.112
Transplantation Proceedings, 45, 2433e2438 (2013)
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by increasing the resistance of liver cells to ischemia and reperfusion events. Ischemic or pharmacological preconditioning interventions that achieve similar effects appear to have the greatest potential to improve clinical outcomes in liver transplantation and liver surgery involving vascular occlusion.2 Pharmacological preconditioning to protect the liver against reperfusion injury has been investigated widely with numerous agents being examined including the following: protease inhibitors,3 glutathione,4 isoflurane,5 and doxorubicin.6 However, the overall benefits for these agents appear to be limited. Octreotide (Oct) is a synthetic somatostatin octapeptide that primarily inhibits growth hormone secretion. Recent studies7e9 have demonstrated that Oct exerts an organ protective effect through several mechanisms including down-regulation of inflammatory mediators, suppression of endotoxin (ETX) levels as well as direct cell protection. Oct has been previously reported to reduce HIRI in a rat isolated hypoxic I/R model.10,11 To further evaluate the possible use of Oct in humans, larger mammal models are required. In the present study, a rabbit HIRI model was used to demonstrate the protective effects of Oct against I/R injury and investigate possible mechanisms of protection. METHODS Animals Preparation Twenty-four New Zealand rabbits of both genders, weighing 1.6e1.9 kg, were used in this study. All animals were obtained from the Laboratory Animal Center of Xiangya Medical School of Central South University. All experimental procedures and study design were reviewed and approved by the Institutional Animal Care Committee of Xiangya Medical School of Central South University, China.
Experimental Protocol All rabbits were fasted for 12 hours prior to the experiment but were given free access to water. Twenty-four rabbits were randomly divided into 3 equal groups: sham operation group (Control), I/R group (I/R), and I/R þ Oct pretreatment group (I/R þ Oct). Each rabbit had anesthesia induced with an intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg). Following the induction of anesthesia, an intravenous (IV) line was established in an ear vein and an isotonic saline solution infusion was begun at 10 mL$kge1$he1. The animal then underwent a tracheotomy and was mechanically ventilated (DH-150 Animal Ventilator, Zhejiang University Medical Apparatus Ltd, Zhejiang, China) on 100% oxygen with a tidal volume of 10 mL/kg. Anesthesia was maintained by a continuous infusion of fentanyl at 10 mg$kge1$he1 and intermittent administrations of 3% pentobarbital (10 mg/kg). Vecuronium was infused at 0.1 mg$kge1$he1 to maintain muscle relaxation. A catheter was inserted into the right carotid artery for blood sampling and systemic hemodynamic measurements. At 30 minutes prior to laparotomy, the I/R þ Oct group rabbits received an injection of Oct 20 mg/kg intraperitoneally and 30 ug/kg subcutaneously (Jilin A-THINK Pharmaceutical Co. Ltd, Jilin, China). Control and I/R group rabbits received the same volume of isotonic saline and all rabbits underwent a laparotomy. HIRI models were set up in the I/R and I/R þ Oct groups using Pringle’s method,12 consisting of 30 minutes of
YANG, SUN, TAKACS ET AL ischemia followed by 120 minutes of reperfusion. Mean arterial pressure (MAP) and heart rate (HR) (HP pressure transducer, HP 78354 Multi-parameter monitor, USA) were measured at various time points: before ischemia (base line, T1), ischemia 15 minutes (T2) and 30 minutes (T3), reperfusion 15 minutes (T4), 30 minutes (T5), 60 minutes (T6), and 120 minutes (T7). Right carotid artery blood samples were drawn and assayed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) (SHIMADZU CL-8000, Japan). Tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b, RI kits, RI Institute of Chinese PLA General Hospital, Bejing, China) levels were assayed at the time intervals T1, T3, T5, T6 and T7. Portal vein blood samples were also drawn and the plasma was analyzed for ETX levels (Endotoxin-specific TAL 1.5 mL/Vial Kit, Xiamen Houshiji, Ltd) at the same time intervals as TNF-a and IL-1b. At the end of the experiment (T7), all animals were humanely killed and the left lobe of the liver was removed. Biopsy specimens of the livers (5mm3) were taken and stored at 80 C for further evaluation of apoptosis using the TUNEL method (TUNEL, TBD2020POD, Boehringer Mannheim Ltd, Germany). A 1 cm3 liver biopsy specimen was also taken from each animal and was fixed in 15% glutaraldehyde to assess Bcl-2 and Bax expression using immunohistochemistry (SABC-FITC kit, SA 1064, Wuhan Boster Biological Technology Ltd.). Another 1 cm3 liver biopsy specimen was also fixed in 2.5% glutaraldehyde phosphate buffer and was stored at 4 C for evaluation of the hepatocellular ultrastructure using electromicroscopy (HITACHI-7500 TEM, Japan).
Statistical Analysis All data are presented as means standard deviation (SD) and the data were analyzed with SPSS for Windows 13.0 statistical software. Differences between groups were analyzed using 1eway analysis of variance (ANOVA) and least-significant difference -q-test. Differences in P values <.05 were considered statistically significant.
RESULTS Hemodynamic Parameters
MAP and HR are shown in Figure 1A and Figure 1B, respectively. Compared with the Control group, significant hemodynamics changes were observed in both I/R groups. However, Oct pretreatment significantly attenuated these changes during I/R (P < .05). Liver Enzymes
A comparison of liver enzymes, ALT, AST, and LDH between groups is shown in Figure 2A, Figure 2B, and Figure 2C. ALT, AST, and LDH were found to be increased in all treatment groups, however, Oct pretreatment significantly decreased their relative levels (P < .05). Inflammatory Cytokine and ETX Level
TNF-a was found to be increased at ischemia 30 minutes in the I/R group and was increased from reperfusion 30 minutes in the I/R þ Oct group (P < .05). IL-1b and ETX levels in I/R and I/R þ Oct groups were increased from ischemia 30 minutes (P < .05). However, the levels were significantly lower in the I/R þ Oct group compared with the I/R group from ischemia 30 minutes (P < .05; Fig 3).
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Fig 1. (A and B) Hemodynamic parameters for each of the 3 groups (mean SD). MAP and HR in the I/R groups were lower than in the control group at ischemia 15 minutes to reperfusion 60 minutes (P < .05). However, the MAP and HR were higher in the I/R þ Oct group compared with those in the I/R group from ischemia 15 minutes to reperfusion 60 minutes (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3 ischemia 15 minutes and 30 minutes respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes respectively.
Apoptosis and Bcl-2, Bax Expression
Liver damage was also confirmed by comparing apoptosis, Bcl-2, and Bax expression at reperfusion 120 minutes (Fig 4; Fig 5). It was noted that Oct pretreatment decreased the rate of apoptosis due to HIRI, increased Bcl-2, and decreased Bax expression (P < .05). Hepatocellular Ultrastructure
Damage to liver cells was directly measured using electromicroscopy (EM). As shown in Figure 6, liver I/R was associated with mitochonridal swelling, mitochondrial ridge fracture, vacuolar degeneration, rough endoplasmic reticulum expansion, structural ambiguity, partial disappearance of nuclear membranes, decrease and even disappearance of glycogen granules, and increase in the number of secondary lysosomes. In contrast, in the Oct pretreatment group, mitochondrial swelling was much less with no significant expansion or degeneration, there was no significant swelling of the endoplasmic reticulum, there were more primary and secondary lysosomes, and the nuclear membrane was more clearly defined. These results would appear to indicate that
Oct pretreament significantly reduced liver cell damage caused by I/R. DISCUSSION
HIRI is a complex, multifactorial pathophysiological process and in liver surgery has been known to significantly affect disease prognosis, surgical success rates, and patient survival.13 Numerous studies have shown that the mechanisms of HIRI include, but are not limited to, excessive intracellular calcium,14 increased free oxygen radicals in activated neutrophils and Kupffer cells,15e19 increased apoptosis in liver parenchymal cells20 and reduction of protective agents such as nitric oxide, heme oxygenase, and heat shock proteins.21,22 In support of previous findings,23,24 our results clearly demonstrated that I/R induced hepatocyte damage. First, I/R significantly increased liver enzymes ALT, AST, and LDH release (Fig 2), which all reflect the degree of hepatocyte damage. Second, I/R caused significant hepatocyte apoptosis (Fig 4 and Fig 5). Finally, at a subcellular level, I/R caused numerous hepatocellular changes, most notably
Fig 2. (A, B, and C) Liver enzymes for each of the 3 groups. The ALT, AST, and LDH levels in the I/R and the I/R þ Oct groups were statistically higher than those in the control group from ischemia 30 minutes to reperfusion 120 minutes (P < .05) with the highest elevation noted at reperfusion 120 minutes. The levels in the I/R þ Oct group, however, were lower than those in the I/R group (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3, ischemia 15 minutes and 30 minutes, respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes, respectively.
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Fig 3. (A, B and C) TNF-a, IL-1b, and ETX levels for each group. TNF-a was increased from ischemia 30 minutes in the I/R group and increased from reperfusion 30 minutes in the I/R þ Oct group (P < .05). IL-1b in the I/R and the I/R þ Oct groups increased from ischemia 30 minutes; both TNF-a and IL-1b showed peak values at reperfusion 60 minutes (P < .05). These levels were, however, significantly lower in the I/R þ Oct group than in the I/R group at these time points (P < .05). The ETX levels in the I/R and the I/R þ Oct groups were higher than in the Control group from ischemia 30 minutes to reperfusion 120 minutes (P < .05); however, it was relatively lower in the I/R þ Oct group than in the I/R group during this period (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3, ischemia 15 minutes and 30 minutes, respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes respectively.
mitochonridal swelling and mitochondrial ridge fracture (Fig 6). Based on our results, we theorize that the mechanisms of hepatocyte damage and systemic effects could be the result of increasing ETX, proinflammatory cytokines TNF-a and IL-1b levels (Fig 3), as well as hepatocyte apoptosis. In addition to clarifying the possible mechanisms of HIRI, we further demonstrated that the agent Oct could attenuate many of the deleterious effects of HIRI resulting in reduced cell damage and destruction. It has been additionally hypothesized that I/R could alter the intestinal microflora and compromise intestinal mucosal barrier function, which could lead to bacterial translocation and endotoxemia.25,26 Indeed, ETX levels were in the
Fig 4. Rates of apoptosis, Bcl-2, and Bax expression for each group. The apoptosis rate for liver cells in the I/R group was significantly greater than was noted in the Control group and the I/R þ Oct group. The I/R þ Oct group, however, had a higher rate of Bcl-2 expression than was noted in the I/R group and the Control group. Finally, Bax expression in the I/R group was noted to be significantly higher than was seen in the Control and the I/R þ Oct groups. * P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct.
normal range during periods of ischemia and apparently increased only after reperfusion due to ETX absorption via the portal vein.27 ETX has also been shown to aggravate liver injury, via altering hepatic energy metabolism and activating Kupffer cells, the latter of which release vasoactive agents and highly active mediators.28,29 These changes can result in a no-reflow phenomenon with the release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, hypoxic cell injury, and hepatocyte apoptosis as an ultimate consequence of endothelial cell damage and hepatic microcirculatory dysfunction, resulting in apparent systemic hemodynamic changes that were noted in our results (Fig 1). Ultimately, endotoxemia has been regarded as a key cause of liver cell injury and systemic damage after I/R. In contrast to these findings, other authors have indicated that most liver cells damaged by I/R actually die as a result of apoptosis.30,31 Bcl-2 is a known antiapoptotic protein32 and its decreased expression is associated with decreased liver cell preservation. We further demonstrated that I/R resulted in an apparent lowering of Bcl-2 levels and higher Bax expression after reperfusion at 120 minutes, supporting the fact that the Bcl-2/Bax apoptosis path is involved in the HIRI. Pharmacological preconditioning has been widely explored as an intervention strategy to overcome the pathophysiological changes caused by HIRI. Studies have demonstrated that pretreatment with natural somatostatin and some if its derivatives (Oct) can improve the hepatocellular energy reserve and significantly reduce LDH and glutamic dehydrogenase (GLDH) release in hypoxic ischemic cell injury in the isolated perfused rat liver.10,33 Pergel et al34 observed the protective effects of somatostatin on HIRI in an ischemic, with or without obstruction, jaundiced rat model, and found that somatostatin was effective in preventing liver injury. Consistent with previous data, our results corroborate that Oct can significantly attenuate hepatocyte damage and hemodynamic changes caused by I/R, as partially noted by a reduction of ALT, AST, and LDH levels in I/R.
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Fig 5. Apoptosis, Bcl-2, and Bax expression. (A, B, and C) The rates of apoptosis cells in each group (A, Control; B, I/R group; C, I/R þ Oct group). (D and E) The rates of Bcl-2 and Bax expression in Control (left), I/R group (middle) and I/R þ Oct (right), respectively (original magnification 400). The arrow indicates Bcl-2 or Bax expression positive cells; nucleus is blue, and the cytoplasm and cell membrane are brown.
Furthermore, cellular evidence was identified showing that treatment with Oct led to an inhibition of apoptosis of heptocytes and preserved their normal morphology as well as decreased the levels of ETX and proinflammatory cytokines such as TNF-a and IL-1b, increased Bcl-2 expression, and decrease Bax expression. Finally, the protective effects of Oct were also supported by electron microscopy at the subcellular levels, showing, among other results, a preserving effect on mitochondrial membranes that may be one of the modes by which Oct exerts its beneficial effects, as mentioned previously, pharmacological inhibition of the mitochondrial transition can protect against reperfusion injury.35,36 Our study had some limitations. Only one dose of Oct (50 mg/kg) was studied. It is unclear whether the protective effect is dose dependent and whether the demonstrated
benefits may increase or decrease with changing doses. Another notable limitation is the fact that this study was conducted in rabbits and the ability to translate these findings to humans is currently unclear, however, beneficial results in a larger mammal model are encouraging. In conclusion, Oct attenuated hemodynamic changes and hepatocyte damage in a rabbit HIRI model. The protective mechanisms of Oct appear to be associated with its ability to decrease the levels of ETX and the proinflammatory cytokines TNF-a and IL-1b, as well as inhibit hepatocellular apoptosis. Based on our animal model and the presented data we suggest that Oct may be useful in decreasing liver injury after liver surgery and/or transplantation and may serve as a promising pharmacological agent for preconditioning against I/R injury.
Fig 6. Liver cell structure using electronmicroscopy in each group. A, Control; B, I/R group; C, I/R þ Oct group. The left arrow indicates mitochondrial vacuolar degeneration; the right arrow indicates mitochondrial ridge fracture.
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REFERENCES 1. Koti RS, Seifalian AM, Davidson BR. Protection of the liver by ischemic preconditioning: a review of mechanisms and clinical applications. Dig Surg. 2003;20:383e396. 2. Jaeschke H. A molecular mechanisms of hepatic ischemiareperfusion injury and preconditioning. JP-Gastrointest Liver Physiol. 2003;284(1):15e26. 3. Kim YI, Hwang YJ, Song KE, et al. Hepatocyte protection by a protease inhibitor against ischemia/reperfusion injury of human liver. J Am Coll Surg. 2002;195:41e50. 4. Liu P, Fisher MA, Farhood A, et al. Beneficial effects of extracellular glutathione against endotoxin-induced liver injury during ischemia and reperfusion. Circ Shock. 1994;43:64e70. 5. Schmidt R, Tritschler E, Hoetzel A, et al. Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury. Ann Surg. 2007;245(6):931e942. 6. Ito K, Ozasa H, Sanada K, et al. Doxorubicin preconditioning: a protection against rat hepatic ischemia-reperfusion injury. Hepatology. 2000;31:416e419. 7. Choi TK, Mok F, Zhan WH, et al. Somatosation in the treatment of acute pancreatitis: a prospective randomized controlled trial. Gut. 1989;30:223e227. 8. Büchler MW, Binder M, Friess H, et al. Potential role of somatostation and octreotide in the management of acute pancreatitis. Digestion. 1994;55(suppl):16e19. 9. Wang XP, Lu Q, Wu JX, et al. The accommodate effect of octreotide on inflammative medium of acute necrotizing pancreatitis. Chin J Internal Med. 2000;3:156e159. 10. Bloechle C, Kusterer K, Konrad T, et al. Rat liver injury induced by hypoxic ischemia and reperfusion: protective action by somatostatins is independent from changes in glucose metabolism. Horm Metab Res. 1994;26(6):270e275. 11. Li JQ, Qi HZ, He ZJ, et al. Protective effect of octreotide on liver warm ischemia reperfusion injury. J Cent South Univ (Med Sci). 2006;31(5):792e796 [in Chinese]. 12. Ito K, Ozasa H, Horikawa S. Effects of prior splenectomy on remnant liver after partial hepatectomy with Pringle maneuver in rats. Liver Int. 2005;25(2):438e444. 13. Arii S, Teramoto K, Kawamura T. Current progress in the understanding of and therapeutic strategies for ischemia and reperfusion injury of the liver. J Hepatobiliary Pancreat Surg. 2003;10(3):189e194. 14. Takemoto Y, Uchida M, Nagasue N, et al. Changes in calcium content of the liver during hepatic ischemia-reperfusion in dogs. Hepatol. 1994;21(5):743e747. 15. Zhou W, Zhang Y, Hosch MS, et al. Subcellular site superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemiadreperfusion. Hepatology. 2001;33(4):902e914. 16. El-Wahsh M, Seifalian AM, Fuller BJ, et al. Induction of adhesion molecule expression in liver ischemia-reperfusion injury is associated with impaired hepatic parenchymal microcirculation. Br J Surg. 2004;91(8):1034e1039. 17. Oikawa K, Ohkohchi N, Sato M, et al. Kupffer cells play an important role in the cytokine production and activation of nuclear factors of liver grafts from non-heart-beating donors. Transplant Int. 2002;15(8):397e405. 18. McCuskey RS. Morphological mechanisms for regulating blood flow through hepatic sinusoids. Liver. 2000;20(1):3e7.
YANG, SUN, TAKACS ET AL 19. Okajima K, Harada N, Kushimoto S, et al. Role of microthrombus formation in the development of ischemia-reperfusioninduced liver injury in rats. Thrombosis Haemostasis. 2002;88(3): 473e480. 20. Harada N, Hatano E, Koizumi N, et al. Akt activation protects rat liver from ischemia-reperfusion injury. J Surg Res. 2004;121(2):159e170. 21. Sakai T, Takaya S, Fukuda A, et al. Evaluation of warm ischemia-reperfusion injury using heat shock protein in the rat liver. Transplant Int. 2003;16(2):88e89. 22. Katori M, Anselmo DM, Busuttil RW, et al. A novel strategy against ischemia and reperfusion injury: Cytoprotection with heme oxygenase system. Transplant Immunol. 2002;9(2e4):227e233. 23. Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol. 2006;290:G1083eG1088. 24. Toledo-Pereyra LH, Suzuki S. Neutrophils, cytokines, and adhesion molecules in hepatic ischemia and reperfusion injury. J Am Coll Surg. 1994;179:758e762. 25. Xing HC, Li LJ, Xu KJ, et al. Intestinal microflora in rats with ischemia/reperfusion liver injury. J Zhejiang Univ SCI. 2005;6B(1):14e21. 26. Ren D, Han D, Zhao Y. Effect of intestinal endotoxemia on hepatic energy metabolism in acute liver failure. Chin J Pathophy. 2001;17(9):890e892 [in Chinese]. 27. Bathe OF, Chow AWC, Phang PT. Splanchnic origin of cytokines in a porcine model of mesenteric ischemia-reperfusion. Surgery. 1998;123:79e88. 28. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury. Am J Surg. 2001;181:160e166. 29. Serrano E, Diaz J, Acosta F, et al. Oxidative stress during ischemia-reperfusion in liver transplantation. Transplant Proc. 2000;32:2651. 30. Cursio R, Gugenheim J, Ricci JE, et al. A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis. FASEB J. 1999;13:253e261. 31. Kohli V, Selzner M, Madden JF, et al. Endothelial cell and hepatocyte deaths occur by apoptosis after ischemia-reperfusion injury in the rat liver. Transplantation. 1999;67:1099e1105. 32. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 1999;399(6735):483e487. 33. Kusterer K, Blochle C, Konrad T, et al. Rat liver injury induced by hypoxic ischemia and reperfusion: protective action by somatostatin and two derivatives. Regul Pept. 1993;44(3): 251e256. 34. Pergel A, Zengin K, Cercel A, et al. The effects of somatostatin and ursodeoxycholic acid in preventing the ischemic injury of the liver following Pringle maneuver in obstructive jaundice-rat model. Hepatogastroenterology. 2007;54(73):229e233. 35. Elimadi A, Sapena R, Settaf A, et al. Attenuation of liver normothermic ischemiaereperfusion injury by preservation of mitochondrial functions with S-15176, a potent trimetazidine derivative. Biochem Pharmacol. 2001;62:509e516. 36. Leducq N, Delmas-Beauvieux MC, Bourdel-Marchasson I, et al. Mitochondrial and energetic dysfunctions of the liver during normothermic reperfusion: protective effect of cyclosporine and role of the mitochondrial permeability transition pore. Transplant Proc. 2000;32:479e480.
D
URING liver surgery surgeons may perform temporary inflow and/or outflow occlusion by vascular clamping to reduce intraoperative blood loss. This technique can lead to hepatic ischemic-reperfusion injury (HIRI) and may sometimes result in multiple system organ failure and/or a systemic inflammatory response syndrome after surgery.1 Due to the risks arising from these complications, therapeutic strategies to reduce HIRI have become a priority. The most promising protective strategy against ischemicreperfusion (I/R) injury explored in the last few years appears to be preconditioning, which presumably functions
From the Department of Anesthesiology of Affiliated Tumor Hospital of Xiang-Ya Medical College of Central South University, Changsha, China (J.Y., H.S., J.L.); Department of Anesthesiology of University of Miami Miller School of Medicine, Miami, FL, USA (P.T., Y.Z., K.A.C.); and Second Xiangya Hospital of Xiangya Medical School, Central South University, Changsha, China (Y.C.). Address reprint requests to Jinfeng Yang, MD, PhD, Department of Anesthesiology of Affiliated Tumor Hospital of Xiang-Ya Medical College of Central South University, 283# Tongzipo Rd., Yuelu Dist., Changsha, China 410013. E-mail: 315977705@qq. com
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.02.112
Transplantation Proceedings, 45, 2433e2438 (2013)
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by increasing the resistance of liver cells to ischemia and reperfusion events. Ischemic or pharmacological preconditioning interventions that achieve similar effects appear to have the greatest potential to improve clinical outcomes in liver transplantation and liver surgery involving vascular occlusion.2 Pharmacological preconditioning to protect the liver against reperfusion injury has been investigated widely with numerous agents being examined including the following: protease inhibitors,3 glutathione,4 isoflurane,5 and doxorubicin.6 However, the overall benefits for these agents appear to be limited. Octreotide (Oct) is a synthetic somatostatin octapeptide that primarily inhibits growth hormone secretion. Recent studies7e9 have demonstrated that Oct exerts an organ protective effect through several mechanisms including down-regulation of inflammatory mediators, suppression of endotoxin (ETX) levels as well as direct cell protection. Oct has been previously reported to reduce HIRI in a rat isolated hypoxic I/R model.10,11 To further evaluate the possible use of Oct in humans, larger mammal models are required. In the present study, a rabbit HIRI model was used to demonstrate the protective effects of Oct against I/R injury and investigate possible mechanisms of protection. METHODS Animals Preparation Twenty-four New Zealand rabbits of both genders, weighing 1.6e1.9 kg, were used in this study. All animals were obtained from the Laboratory Animal Center of Xiangya Medical School of Central South University. All experimental procedures and study design were reviewed and approved by the Institutional Animal Care Committee of Xiangya Medical School of Central South University, China.
Experimental Protocol All rabbits were fasted for 12 hours prior to the experiment but were given free access to water. Twenty-four rabbits were randomly divided into 3 equal groups: sham operation group (Control), I/R group (I/R), and I/R þ Oct pretreatment group (I/R þ Oct). Each rabbit had anesthesia induced with an intraperitoneal injection of 3% sodium pentobarbital (30 mg/kg). Following the induction of anesthesia, an intravenous (IV) line was established in an ear vein and an isotonic saline solution infusion was begun at 10 mL$kge1$he1. The animal then underwent a tracheotomy and was mechanically ventilated (DH-150 Animal Ventilator, Zhejiang University Medical Apparatus Ltd, Zhejiang, China) on 100% oxygen with a tidal volume of 10 mL/kg. Anesthesia was maintained by a continuous infusion of fentanyl at 10 mg$kge1$he1 and intermittent administrations of 3% pentobarbital (10 mg/kg). Vecuronium was infused at 0.1 mg$kge1$he1 to maintain muscle relaxation. A catheter was inserted into the right carotid artery for blood sampling and systemic hemodynamic measurements. At 30 minutes prior to laparotomy, the I/R þ Oct group rabbits received an injection of Oct 20 mg/kg intraperitoneally and 30 ug/kg subcutaneously (Jilin A-THINK Pharmaceutical Co. Ltd, Jilin, China). Control and I/R group rabbits received the same volume of isotonic saline and all rabbits underwent a laparotomy. HIRI models were set up in the I/R and I/R þ Oct groups using Pringle’s method,12 consisting of 30 minutes of
YANG, SUN, TAKACS ET AL ischemia followed by 120 minutes of reperfusion. Mean arterial pressure (MAP) and heart rate (HR) (HP pressure transducer, HP 78354 Multi-parameter monitor, USA) were measured at various time points: before ischemia (base line, T1), ischemia 15 minutes (T2) and 30 minutes (T3), reperfusion 15 minutes (T4), 30 minutes (T5), 60 minutes (T6), and 120 minutes (T7). Right carotid artery blood samples were drawn and assayed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) (SHIMADZU CL-8000, Japan). Tumor necrosis factor a (TNF-a) and interleukin 1b (IL-1b, RI kits, RI Institute of Chinese PLA General Hospital, Bejing, China) levels were assayed at the time intervals T1, T3, T5, T6 and T7. Portal vein blood samples were also drawn and the plasma was analyzed for ETX levels (Endotoxin-specific TAL 1.5 mL/Vial Kit, Xiamen Houshiji, Ltd) at the same time intervals as TNF-a and IL-1b. At the end of the experiment (T7), all animals were humanely killed and the left lobe of the liver was removed. Biopsy specimens of the livers (5mm3) were taken and stored at 80 C for further evaluation of apoptosis using the TUNEL method (TUNEL, TBD2020POD, Boehringer Mannheim Ltd, Germany). A 1 cm3 liver biopsy specimen was also taken from each animal and was fixed in 15% glutaraldehyde to assess Bcl-2 and Bax expression using immunohistochemistry (SABC-FITC kit, SA 1064, Wuhan Boster Biological Technology Ltd.). Another 1 cm3 liver biopsy specimen was also fixed in 2.5% glutaraldehyde phosphate buffer and was stored at 4 C for evaluation of the hepatocellular ultrastructure using electromicroscopy (HITACHI-7500 TEM, Japan).
Statistical Analysis All data are presented as means standard deviation (SD) and the data were analyzed with SPSS for Windows 13.0 statistical software. Differences between groups were analyzed using 1eway analysis of variance (ANOVA) and least-significant difference -q-test. Differences in P values <.05 were considered statistically significant.
RESULTS Hemodynamic Parameters
MAP and HR are shown in Figure 1A and Figure 1B, respectively. Compared with the Control group, significant hemodynamics changes were observed in both I/R groups. However, Oct pretreatment significantly attenuated these changes during I/R (P < .05). Liver Enzymes
A comparison of liver enzymes, ALT, AST, and LDH between groups is shown in Figure 2A, Figure 2B, and Figure 2C. ALT, AST, and LDH were found to be increased in all treatment groups, however, Oct pretreatment significantly decreased their relative levels (P < .05). Inflammatory Cytokine and ETX Level
TNF-a was found to be increased at ischemia 30 minutes in the I/R group and was increased from reperfusion 30 minutes in the I/R þ Oct group (P < .05). IL-1b and ETX levels in I/R and I/R þ Oct groups were increased from ischemia 30 minutes (P < .05). However, the levels were significantly lower in the I/R þ Oct group compared with the I/R group from ischemia 30 minutes (P < .05; Fig 3).
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Fig 1. (A and B) Hemodynamic parameters for each of the 3 groups (mean SD). MAP and HR in the I/R groups were lower than in the control group at ischemia 15 minutes to reperfusion 60 minutes (P < .05). However, the MAP and HR were higher in the I/R þ Oct group compared with those in the I/R group from ischemia 15 minutes to reperfusion 60 minutes (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3 ischemia 15 minutes and 30 minutes respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes respectively.
Apoptosis and Bcl-2, Bax Expression
Liver damage was also confirmed by comparing apoptosis, Bcl-2, and Bax expression at reperfusion 120 minutes (Fig 4; Fig 5). It was noted that Oct pretreatment decreased the rate of apoptosis due to HIRI, increased Bcl-2, and decreased Bax expression (P < .05). Hepatocellular Ultrastructure
Damage to liver cells was directly measured using electromicroscopy (EM). As shown in Figure 6, liver I/R was associated with mitochonridal swelling, mitochondrial ridge fracture, vacuolar degeneration, rough endoplasmic reticulum expansion, structural ambiguity, partial disappearance of nuclear membranes, decrease and even disappearance of glycogen granules, and increase in the number of secondary lysosomes. In contrast, in the Oct pretreatment group, mitochondrial swelling was much less with no significant expansion or degeneration, there was no significant swelling of the endoplasmic reticulum, there were more primary and secondary lysosomes, and the nuclear membrane was more clearly defined. These results would appear to indicate that
Oct pretreament significantly reduced liver cell damage caused by I/R. DISCUSSION
HIRI is a complex, multifactorial pathophysiological process and in liver surgery has been known to significantly affect disease prognosis, surgical success rates, and patient survival.13 Numerous studies have shown that the mechanisms of HIRI include, but are not limited to, excessive intracellular calcium,14 increased free oxygen radicals in activated neutrophils and Kupffer cells,15e19 increased apoptosis in liver parenchymal cells20 and reduction of protective agents such as nitric oxide, heme oxygenase, and heat shock proteins.21,22 In support of previous findings,23,24 our results clearly demonstrated that I/R induced hepatocyte damage. First, I/R significantly increased liver enzymes ALT, AST, and LDH release (Fig 2), which all reflect the degree of hepatocyte damage. Second, I/R caused significant hepatocyte apoptosis (Fig 4 and Fig 5). Finally, at a subcellular level, I/R caused numerous hepatocellular changes, most notably
Fig 2. (A, B, and C) Liver enzymes for each of the 3 groups. The ALT, AST, and LDH levels in the I/R and the I/R þ Oct groups were statistically higher than those in the control group from ischemia 30 minutes to reperfusion 120 minutes (P < .05) with the highest elevation noted at reperfusion 120 minutes. The levels in the I/R þ Oct group, however, were lower than those in the I/R group (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3, ischemia 15 minutes and 30 minutes, respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes, respectively.
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Fig 3. (A, B and C) TNF-a, IL-1b, and ETX levels for each group. TNF-a was increased from ischemia 30 minutes in the I/R group and increased from reperfusion 30 minutes in the I/R þ Oct group (P < .05). IL-1b in the I/R and the I/R þ Oct groups increased from ischemia 30 minutes; both TNF-a and IL-1b showed peak values at reperfusion 60 minutes (P < .05). These levels were, however, significantly lower in the I/R þ Oct group than in the I/R group at these time points (P < .05). The ETX levels in the I/R and the I/R þ Oct groups were higher than in the Control group from ischemia 30 minutes to reperfusion 120 minutes (P < .05); however, it was relatively lower in the I/R þ Oct group than in the I/R group during this period (P < .05). *P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct. T1, base line; T2 and T3, ischemia 15 minutes and 30 minutes, respectively; T4, T5, T6, and T7, reperfusion 15 minutes, 30 minutes, 60 minutes, and 120 minutes respectively.
mitochonridal swelling and mitochondrial ridge fracture (Fig 6). Based on our results, we theorize that the mechanisms of hepatocyte damage and systemic effects could be the result of increasing ETX, proinflammatory cytokines TNF-a and IL-1b levels (Fig 3), as well as hepatocyte apoptosis. In addition to clarifying the possible mechanisms of HIRI, we further demonstrated that the agent Oct could attenuate many of the deleterious effects of HIRI resulting in reduced cell damage and destruction. It has been additionally hypothesized that I/R could alter the intestinal microflora and compromise intestinal mucosal barrier function, which could lead to bacterial translocation and endotoxemia.25,26 Indeed, ETX levels were in the
Fig 4. Rates of apoptosis, Bcl-2, and Bax expression for each group. The apoptosis rate for liver cells in the I/R group was significantly greater than was noted in the Control group and the I/R þ Oct group. The I/R þ Oct group, however, had a higher rate of Bcl-2 expression than was noted in the I/R group and the Control group. Finally, Bax expression in the I/R group was noted to be significantly higher than was seen in the Control and the I/R þ Oct groups. * P < .05 vs Control group; þP < .05 I/R group vs I/R þ Oct.
normal range during periods of ischemia and apparently increased only after reperfusion due to ETX absorption via the portal vein.27 ETX has also been shown to aggravate liver injury, via altering hepatic energy metabolism and activating Kupffer cells, the latter of which release vasoactive agents and highly active mediators.28,29 These changes can result in a no-reflow phenomenon with the release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, hypoxic cell injury, and hepatocyte apoptosis as an ultimate consequence of endothelial cell damage and hepatic microcirculatory dysfunction, resulting in apparent systemic hemodynamic changes that were noted in our results (Fig 1). Ultimately, endotoxemia has been regarded as a key cause of liver cell injury and systemic damage after I/R. In contrast to these findings, other authors have indicated that most liver cells damaged by I/R actually die as a result of apoptosis.30,31 Bcl-2 is a known antiapoptotic protein32 and its decreased expression is associated with decreased liver cell preservation. We further demonstrated that I/R resulted in an apparent lowering of Bcl-2 levels and higher Bax expression after reperfusion at 120 minutes, supporting the fact that the Bcl-2/Bax apoptosis path is involved in the HIRI. Pharmacological preconditioning has been widely explored as an intervention strategy to overcome the pathophysiological changes caused by HIRI. Studies have demonstrated that pretreatment with natural somatostatin and some if its derivatives (Oct) can improve the hepatocellular energy reserve and significantly reduce LDH and glutamic dehydrogenase (GLDH) release in hypoxic ischemic cell injury in the isolated perfused rat liver.10,33 Pergel et al34 observed the protective effects of somatostatin on HIRI in an ischemic, with or without obstruction, jaundiced rat model, and found that somatostatin was effective in preventing liver injury. Consistent with previous data, our results corroborate that Oct can significantly attenuate hepatocyte damage and hemodynamic changes caused by I/R, as partially noted by a reduction of ALT, AST, and LDH levels in I/R.
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Fig 5. Apoptosis, Bcl-2, and Bax expression. (A, B, and C) The rates of apoptosis cells in each group (A, Control; B, I/R group; C, I/R þ Oct group). (D and E) The rates of Bcl-2 and Bax expression in Control (left), I/R group (middle) and I/R þ Oct (right), respectively (original magnification 400). The arrow indicates Bcl-2 or Bax expression positive cells; nucleus is blue, and the cytoplasm and cell membrane are brown.
Furthermore, cellular evidence was identified showing that treatment with Oct led to an inhibition of apoptosis of heptocytes and preserved their normal morphology as well as decreased the levels of ETX and proinflammatory cytokines such as TNF-a and IL-1b, increased Bcl-2 expression, and decrease Bax expression. Finally, the protective effects of Oct were also supported by electron microscopy at the subcellular levels, showing, among other results, a preserving effect on mitochondrial membranes that may be one of the modes by which Oct exerts its beneficial effects, as mentioned previously, pharmacological inhibition of the mitochondrial transition can protect against reperfusion injury.35,36 Our study had some limitations. Only one dose of Oct (50 mg/kg) was studied. It is unclear whether the protective effect is dose dependent and whether the demonstrated
benefits may increase or decrease with changing doses. Another notable limitation is the fact that this study was conducted in rabbits and the ability to translate these findings to humans is currently unclear, however, beneficial results in a larger mammal model are encouraging. In conclusion, Oct attenuated hemodynamic changes and hepatocyte damage in a rabbit HIRI model. The protective mechanisms of Oct appear to be associated with its ability to decrease the levels of ETX and the proinflammatory cytokines TNF-a and IL-1b, as well as inhibit hepatocellular apoptosis. Based on our animal model and the presented data we suggest that Oct may be useful in decreasing liver injury after liver surgery and/or transplantation and may serve as a promising pharmacological agent for preconditioning against I/R injury.
Fig 6. Liver cell structure using electronmicroscopy in each group. A, Control; B, I/R group; C, I/R þ Oct group. The left arrow indicates mitochondrial vacuolar degeneration; the right arrow indicates mitochondrial ridge fracture.
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