Year : 2009 | Volume
: 20 | Issue : 6 | Page : 1010--1014
Reduction of the severity of ischemia reperfusion-induced pancreatitis by ischemic pre-conditioning of the liver
Saman Nikeghbalian, Mohsen Reza Mansoorian, Seyed Mohammad Vahid Hosseini, Parviz Mardani, Bita Geramizadeh, Seyed Ali Malek Hosseini
Department of Surgery, University of Shiraz Medical Sciences and Health Services, Transplantation Research Centre, Shiraz, Iran
Seyed Mohammad Vahid Hosseini
Department of Surgery, University of Shiraz Medical Sciences and Health Services, Shiraz
Pre-conditioning by brief exposure to ischemia does not only protect the concerned organ against subsequent severe ischemic damage, but also has protective effect on other organs, which is called remote pre-conditioning. Our aim in this study was to evaluate the protective effect of brief liver ischemia on the pancreas against severe ischemia-reperfusion-induced pancreatitis. This study was performed on 30 male wistar rats. Ischemic pre-conditioning of liver was performed by first clamping of the hepatic pedicle for 10 minutes. Following this, ischemia-reperfusion of the pancreas was performed by first clam-ping the inferior splenic artery for 30 minutes, followed by reperfusion for one hour. The rats were divided into three groups (10 rats in each group). GroupOne was the sham operated group, without clamping of any artery. Group-two developed ischemiareperfusion-induced pancreatitis, without ischemic pre-conditioning of the liver, while Group-three underwent ischemic pre-conditioning of the liver followed by ischemia-reperfusion of the pancreas. Ischemic pre-conditioning, applied prior to induction of pancreatitis, caused a reduction in plasma lipase, plasma interleukin-1β and histological signs of pancreatic damage, but plasma interleukin-10 levels were not significantly different between the three groups. Ischemic pre-conditioning of the liver did not cause any alteration of the liver enzymes. Our study suggests that ischemic preconditioning of the liver reduces the severity of ischemia-reperfusion-induced pancreatitis. These effects are partly related to the reduction of pro-inflammatory interleukin -1β.
|How to cite this article:|
Nikeghbalian S, Mansoorian MR, Hosseini SV, Mardani P, Geramizadeh B, Hosseini SM. Reduction of the severity of ischemia reperfusion-induced pancreatitis by ischemic pre-conditioning of the liver.Saudi J Kidney Dis Transpl 2009;20:1010-1014
|How to cite this URL:|
Nikeghbalian S, Mansoorian MR, Hosseini SV, Mardani P, Geramizadeh B, Hosseini SM. Reduction of the severity of ischemia reperfusion-induced pancreatitis by ischemic pre-conditioning of the liver. Saudi J Kidney Dis Transpl [serial online] 2009 [cited 2020 Aug 3 ];20:1010-1014
Available from: http://www.sjkdt.org/text.asp?2009/20/6/1010/57255
Acute pancreatitis is caused by premature active enzymes, but a disturbance of pancreatic blood flow is also involved in its pathophysiology, which leads to formation of thrombi in capillaries and release of oxygen-derived free radicals and pro-inflammatory cytokines. ,
Many organs respond to brief exposure to ischemia with an increase in resistance to prevent severe ischemia, which is called ischemic preconditioning. , Ischemic pre-conditioning was first discovered by Murry et al in a canine model of regional myocardial ischemia.  The ischemic pre-conditioning attenuates formation of apoptosis of pancreatic tissue in warm ischemia during rejection of human pancreas allografts as well as in experimental pancreatic ischemia reperfusion injury. ,,
Studies of the effects of ischemic pre-conditioning on distant organs may help clarify its mechanism of action. Consequently, we undertook a study to determine whether a period of brief liver ischemia, which is known to be locally protective, will also protect the pancreas.
Materials and Methods
All experiments were performed in the animal laboratory and transplantation research center. The University Ethics Committee approved this study. This study was performed on 30 male wistar rats, each weighing 200-220 grams all of whom were fasted for 18 hours prior to experimentation; they had free access to drinking water up to six hours prior to experimentation.
All the study rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg). After the abdominal wall was cleaned with 10% povidine iodine (betadine), the abdomen was opened by a midline incision.
The study animals were divided into three groups for the purpose of the study, each group containing 10 animals, as follows:
Group-1. Sham operated control group : In this group, after the first laparotomy, mobilization of the pancreas without clamping any arteries was performed and the incision was closed after 30 minutes. One hour later, re-exploration and sampling were done.
Group-2. Rats with ischemia-reperfusion pancreatitis : In this group, clamping of splenic inferior artery was performed with micro vascular clamp for 30 minutes following which the clamp was removed and abdomen was closed. One hour later and after reperfusion, re-exploration was done for sampling.
Group-3. Rats exposed to liver ischemic preconditioning : In this group, ischemic preconditioning was achieved by brief occlusion of the hepatic pedicle with a microvascular clamp for 10 minutes, followed by 10 minutes of reperfusion. This was done before undertaking 30 minutes of pancreatic ischemia followed by reperfusion for one hour.
Blood samples were taken about one hour after termination of first laparotomy. The samples were taken from the aorta under general anesthesia before the animals were killed. Samples were centrifuged and stored at -30° Celcius and sent for pathological examination. Plasma lipase activity was determined with a Kodak Ectashem DT II system analyzer. The values of plasma lipase activity were expressed as units/liter. Plasma IL-1B and IL-10 were measured using the Bio source cystoscreen rat IL-IB and IL-10 kits based on ELISA. Concentration of interleukin was expressed as pg/mL.
Serum levels of aspartate aminotransferase (AST), alanine aminotransfrase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gama glutamyl transferase (GGTP) were determined with a Hitachi 911 automatic analyzer as Units/L.
Samples of tissue excised from the body portion of the pancreas were fixed for 24 hours in buffered 10% formalin, embedded in paraffin, and sections were stained with hematoxylin and eosin. The histological grading of edema was made using a scale ranging from 0 to 3 (0 = no edema, 1 = interlobular edema, 2 = interlobular and moderate intralobular edema, and 3 = interlobular edema and severe intralobular edema). Leukocytic infiltration was also graded from 0 to 3 (0 = absent, 1 = scarce perivascular infiltration, 2 = moderate perivascular and scarce diffuse infiltration, 3 = abundant diffuse infiltration). Grading of vacuolization was based on the appropriate percentage of acinar cells involved: 0 = absent, 1 = less than 25%, 2 = 2550% and 3 = more than 50% of acinar cells. Hemorrhage was graded as: 0 = no hemorrhage, 1 = 1-2 hemorrhagic foci per slide, 2 = 3-5 hemorrhagic foci per slide, 3 = more than 5 hemorrhagic foci per slide. Necrosis was graded as: 0 = no necrosis, 1= less than 15% of pancreatic cells involved, 2 = 15-35% of cells involved, 3 = more than 35% of cells involved. ,,,,
The differences between the mean values from various groups of experiments were compared by variance analysis and Student's t-test for unpaired data. A difference with a P value of less than 0.05 was considered statistically significant. Results are expressed as means ± S.E.M.
The pancreas of the sham operated animals (Group 1) showed, both macroscopically and on light microscopy, no tissue alteration in 70% of the rats. Only 30% of rats showed mild interlobular edema, 10% of whom showed one to two foci of hemorrhage per slide.
Exposure to ischemic pre-conditioning (Group 3) as against ischemia-reperfusion alone (Group 2) caused mild interlobular edema in 60% and moderate intralobular and interlobular edema in 10% of the rats. Forty percent of rats in this group showed one to two foci of hemorrhage but there was no histological evidence of leukocyte infiltration, necrosis and/or vacuolization.
Pancreatic ischemia for 30 minutes followed by one hour of reperfusion (Group 2) produced interlobular edema in 80% and moderate intralobular and interlobular edema in 20% of the rats. Sixty percent of rats in this group showed one to two foci of hemorrhage and 60% showed mild perivascular leukocyte infiltration and 20% showed moderate perivascular and mild diffuse infiltration, in addition. In 10% of the rats, pancreatic necrosis was seen due to ischemia-reperfusion. None of them showed vacuolization [Table 1].
Plasma lipase activity
Plasma lipase activity in Group-1 rats (control group) was 15.7 ± 1.7 U/L. Ischemic pre-conditioning followed by ischemia-reperfusion did not significantly affect the plasma lipase activity. Ischemia-reperfusion-induced pancreatitis caused an increase in plasma lipase activity to 32.4 ± 12.9 U/L. Thus, ischemic pre-conditioning strongly and significantly reduced ischemia-reperfusionevoked increase in plasma lipase activity in the study animals (P = 0.005).
Plasma interleukin-1β and interleukin-10 concentration
In control sham-operated rats, plasma interleukin-1β concentration was 28.6 ± 17 pg/mL. Ischemia-reperfusion-induced pancreatitis caused an increase in plasma interleukin-1(3 to 58.8 ± 12.3 pg/mL. In rats that were subject to ischemic pre-conditioning, this parameter was 47.7 ± 9.5 pg/mL. Thus, ischemic pre-conditioning significantly reduced the ischemia-reperfusionevoked increase in plasma interleukin-1β concentration compared with the ischemia-reperfusion group (P = 0.037).
In control sham-operated rats, plasma interleukin-10 concentration was 0.000 pg/mL and in rats which underwent ischemic pre-conditioning before ischemia-reperfusion, it was 31.6 ± 42.9 pg/mL. The interleukin-10 concentration was 50.01 ± 47.03 pg/mL in the ischemia-reperfusion group without ischemic pre-conditioning. Thus ischemic pre-conditioning did not significantly reduce the ischemia-reperfusion-evoked increase in plasma interleukin-10 concentration (P = 0.374).
Ten minutes of hepatic ischemia and then reperfusion of liver did not cause any difference in the liver enzymes compared to the sham group. Thus, ischemic pre-conditioning of the liver had no significant effect on liver parenchymal function.
The locally protective effect of ischemic preconditioning is well known. ,, Recently, it has been suggested that ischemic pre-conditioning also has a distant effect. ,, In this study, we attempted to determine whether brief hepatic ischemia, known to be effective locally, has a remote effect on ischemia-reperfusion injury to the pancreas.
The protective effect of ischemic pre-conditioning against lesions caused by subsequent severe ischemia was first described in the heart by Murry et al.  Dembinski A et al showed that ischemic pre-conditioning, applied prior to ischemia-reperfusion-induced pancreatitis, strongly reduces the severity of acute pancreatitis. The beneficial effect of ischemic pre-conditioning in the pancreas was manifested by a reduction in plasma lipase activity and a decrease in plasma concentration of pro-inflammatory interleukin-1β .  They found a close relationship between pancreatitis evoked by ischemic pre-conditioning and a decrease in biochemical signs of pancreatitis as well as the improvement of pancreatic blood flow and reduction in histological score of pancreatic damage. 
Activation of leukocytes and release of proinflammatory cytokines are responsible for causing local pancreatic damage and development of systemic inflammatory response syndrome and multiple organ failure in the course of acute pancreatitis.  Pro-inflammatory cytokines such as IL-1(3, IL-6 and tumor necrosis factor-α (TNFα) are produced within the pancreas and subsequently within distant organs, that develop organ dysfunction during severe pancreatitis. 
Anti-inflammatory and healing-promoting effect of ischemic pre-conditioning seems to be also related to the release of IL-10, which has been found to be a major anti-inflammatory cytokine. 
Inflammatory infiltration plays an important role in development of pancreatic damage in the course of acute pancreatitis. Leukocytes adhere to the vascular endothelium forming plagues and contribute to the injury by reducing blood flow via occlusion of microvessels and release of mediators of tissue damage. The release of interleukin-10 plays a crucial role in the release of other members of the pro-inflammatory cytokine cascade and activates the systemic acute phase of inflammation.  These observations are in agreement with our present data and partly elucidate the mechanism of protective effect after ischemic pre-conditioning. In our present study, ischemic pre-conditioning reduced the leukocyte infiltration of pancreatic tissue and inhibited the production of interleukin-10 leading to the reduction of pancreatic damage. Dembinski A et al have shown earlier that there is no alteration of plasma interleukin-10 concentration after ischemia-reperfusion-induced pancreatitis or ischemic pre-conditioning applied alone or in combination, with induction of acute pancreatitis.  However, in another study in 2006, they showed that ischemic pre-conditioning increases release of interleukin-10. 
In our study, in animals subjected to pancreatic ischemia-reperfusion without ischemic preconditioning, we found that plasma lipase activity and interleukin 1-β concentration were significantly higher than in animals with ischemic pre-conditioning, and interleukin-10 concentration was lower, but only marginally.
Our study suggests that morphological alteration of acute pancreatitis was less in animals that underwent ischemic pre-conditioning prior to ischemia-reperfusion.
Our study shows that brief hepatic ischemia and reperfusion had a beneficial remote effect on the pancreas. This finding was confirmed by biochemical and histological studies. We suggest that treatment of cadaveric livers with ischemic pre-conditioning prior to procurement would protect the pancreas as well.
The authors would like to thank Dr. Babak Sabet for helping in statistical analysis
|1||Dembinski A, Warzecha Z, Ceranowicz P, Tomaszewska R, Dembinski M, Pabianczyk M. Ischemic preconditioning reduces the severity of ischemia/reperfusion-induced pancreatitis. Eur J Pharmacol 2003;473:207-16.|
|2||Geroge H, Gregory G, Michael G. Ischemia/ reperfusion-induced pancreatitis. Dig Surg 2000; 17:3-17.|
|3||Rock P, Yao Z. Ischemia/reperfusion injury, preconditioning and critical illness. Curr Opin Anaesthesiol 2002;15:139-46.|
|4||Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.|
|5||Drognitz O, Liu X, Obermier R, Neeff H. Ischemic preconditioning fails to improve microcirculation but increases apoptetic cell death in experimental pancreas transplantation. Transpl Int 2004;17:317-24.|
|6||Ates E, Genc E, Erdasap N, Erkasaps, Akman S, Firat P. Renal protection by brief liver ischemia in rats. Transplantation 2002;74:1247-51.|
|7||Gho BC, Schoemaker RG, Van Den Doel MA, et al. Myocardial protection by brief ischemia in non cardiac tissue. Circulation 1996;94:2193-200.|
|8||Peralta C, Prats N, Xaus C, et al. Protective effect of liver ischemic preconditioning on liver and lung injury induced by hepatic ischemia/ reperfusion in the rat. Hepatology 1999;30: 1481-9.|
|9||Dembinski A, Warzecha Z, Ceranowicz P, et al. effect of ischemic preconditioning on pancreatic regeneration and pancreatic expression of vascular endothelial growth factor and platelet derived growth factor-A in ischemia/reperfusioninduced pancreatitis. Physiol Pharmacol 2006; 57(1):39-58.|