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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
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


Department of Surgery, University of Shiraz Medical Sciences and Health Services, Transplantation Research Centre, Shiraz, Iran

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Date of Web Publication27-Oct-2009
 

   Abstract 

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;20:1010-4

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 2019 Dec 6];20:1010-4. Available from: http://www.sjkdt.org/text.asp?2009/20/6/1010/57255

   Introduction Top


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. [1],[2]

Many organs respond to brief exposure to is­chemia with an increase in resistance to prevent severe ischemia, which is called ischemic pre­conditioning. [1],[3] Ischemic pre-conditioning was first discovered by Murry et al in a canine model of regional myocardial ischemia. [4] 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. [5],[6],[7]

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 Top


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. [1],[2],[3],[4],[5]


   Statistical Analysis Top


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.


   Results Top


Morphological features

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).

Liver Enzymes

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.


   Discussion Top


The locally protective effect of ischemic pre­conditioning is well known. [1],[2],[3] Recently, it has been suggested that ischemic pre-conditioning also has a distant effect. [6],[7],[8] 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. [4] 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 . [1] 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. [1]

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. [9] 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. [9]

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. [9]

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. [1] 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. [1] However, in another study in 2006, they showed that ischemic pre-conditioning in­creases release of interleukin-10. [9]

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.


   Conclusion Top


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.


   Acknowledgement Top


The authors would like to thank Dr. Babak Sabet for helping in statistical analysis

 
   References Top

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.  Back to cited text no. 1      
2.Geroge H, Gregory G, Michael G. Ischemia/ reperfusion-induced pancreatitis. Dig Surg 2000; 17:3-17.  Back to cited text no. 2      
3.Rock P, Yao Z. Ischemia/reperfusion injury, pre­conditioning and critical illness. Curr Opin Anaesthesiol 2002;15:139-46.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
4.Murry CE, Jennings RB, Reimer KA. Precondi­tioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124-36.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
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.  Back to cited text no. 5      
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.  Back to cited text no. 6      
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.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
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.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  
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.  Back to cited text no. 9      

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Correspondence Address:
Seyed Mohammad Vahid Hosseini
Department of Surgery, University of Shiraz Medical Sciences and Health Services, Shiraz
Iran
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PMID: 19861862

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This article has been cited by
1 Protection of organs other than the heart by remote ischemic conditioning
Candilio, L. and Malik, A. and Hausenloy, D.J.
Journal of Cardiovascular Medicine. 2013; 14(3): 193-205
[Pubmed]



 

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    Abstract
    Introduction
    Materials and Me...
    Statistical Analysis
    Results
    Discussion
    Conclusion
    Acknowledgement
    References
    Article Tables
 

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