Chun-Hui Yuan, Gui-Chen Li, He Zhang, Ying Cheng, Ning Zhao, Yong-Feng Liu, Department of Organ Transplantation, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
Supported by a fund of Liaoning Province key Project, No.0025001
Correspondence to: Dr. Chun-Hui Yuan, Department of Organ Transplantation, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Provience, China.  ychdoctor@hotmail.com
Telephone: +86-24-23256666    Fax: +86-24-23265284
Received: 2003-06-05    Accepted: 2003-11-06

Abstract
AIM: To evaluate the viability and energy metabolism of long warm ischemically damaged pancreas during preservation by the UW solution cold storage method.

METHODS: The pancreas grafts subjected to 30-120 min warm ischemia were preserved by the UW solution cold storage method for 24 h. The tissue concentrations of adenine nucleotides (AN) and adenosine triphosphate (ATP) and total adenine nucleotides (TAN)  were determined by using high performance liquid chromatography (HPLC) and the viability of the pancreas graft was tested in the canine model of segmental pancreas autotransplantation.

RESULTS: The functional success rates of pancreas grafts of groups after 30 min, 60 min, 90 min, 120 min of warm ischemia were 100%, 100%, 67.7%, 0%, respectively. There was an excellent correlation between the posttransplant viability and tissue concentration of ATP and TAN at the end of preservation.

CONCLUSION: The UW solution cold storage method was effective for functional recovery of the pancreas suffering 60-min warm ischemia. The tissue concentration of ATP and TAN at the end of 24 h preservation by the UW solution cold storage method would predict the posttransplant outcome of pancreas graft subjected to significant warm ischemia.

Yuan CH, Li GC, Zhang H, Cheng Y, Zhao N, Liu YF. Evaluation of the viability and energy metabolism of canine pancreas graft subjected to significant warm ischemia damage during preservation by UW solution cold storage method. World J Gastroenterol  2004; 10(12): 1785-1788
http://www.wjgnet.com/1007-9327/10/1785.asp

INTRODUCTION
Preservation is necessary if organs for transplantation are removed from donors prior to preparation of the recipient^[1-3]. The methods used for preservation of pancreas grafts for experimental transplantation have produced variable results^[4,5]. As warm ischemic injury of pancreas graft before and during procurement strongly influences the results of pancreas transplantation, it is important to predict the viability of the ischemically damaged pancreas graft before transplantation^[6,7]. Recently we preserved the segmental pancreas in the UW solution after 30-120 min, and our experimental model was heterotopic segmental (left limb) pancreas autotransplantation in totally pancreatectomized dogs, and demonstrated that the UW solution cold storage method was effective for functional recovery of the pancreas suffering 60-min warm ischemia^[8]. There are some qualitative differences between warm and cold ischemic injuries^[9-12]. In this study we examined the viability and energy metabolism of the pancreas graft after significant warm ischemia and cold storage, and found tissue concentrations of ATP and TAN after preservation by the UW solution cold storage method were excellent markers to predict the posttransplant outcome^[13,14] .

MATERIALS AND METHODS
Animals
Mongrel dogs of both sexes, weighing 10-15 kg were used for the experiments. UW solution was from China Pharmaceutical Corporation Guangzhou Branch. Chemicals were from Sigma Co.Ltd.
      Operative procedures are as follows. Anesthesia was induced and maintained with sodium pentobarbiturate (25 mg/kg ). The pancreas was exposed through a midline abdominal incision, and the left limb (tail) was removed with the splenic vessels in preparation for grafting, followed by splenectomy. The segmental pancreas graft was unflushed and left in situ for 30-120 min. After warm ischemia the pancreas was flushed with 30-50 mL heparinized cold UW solution (10 000 units/L heparin) and preserved in 50 mL heparinized cold UW solution for 24 h. A splenectomy was performed. The remainder of the pancreas was excised at the time of transplantation. The pancreatic tail was autotransplanted heterotopically, immediately or after preservation, with anastomosis of the splenic vessels to iliac vessels. A proper delicate tube was inserted into the pancreatic duct to drain the pancreatic juice. After operation , the dogs received saline with 100 g/L glucose (30 mL /kg) and 3.2 Mu penicillin for 3-5 days, then standard kennel diets were given.

Experimental protocol
There were two groups of control dogs: group 1, sham-operated group, abdomen was only opened and closed; group 2, no warm ischemia, pancreatic tail was flushed and preserved immediately after being harvested. The experimental group (group 3) was divided into 4 subgroups according to the warm ischemia time: group 3a, 30 min warm ischemia; group 3b, 60 min warm ischemia; group 3c, 90 min warm ischemia; group 3d, 120 min warm ischemia.

Functional studies
Blood glucose concentration was determined daily during the first postoperative week after autotransplantation. An intravenous glucose tolerance test (IVGTT) was performed one week after transplantation. Glucose, 0.5 g/kg, was administered as a bolus and blood samples were collected serially over a 2-h period for plasma glucose. IVGTT K values were calculated from the plasma glucose levels at the end of 5 to 60 min^[15]. Maintenance of normoglycemia for at least five days after transplantation or a key value of IVGTT more than 1.0 one week after transplantation was considered a functional success of pancreas graft. The plasma insulin levels in splenic and peripheral vein one hour after transplantation were examined. The pancreatic juice was collected every day and amylopsin in the pancreatic juice of the first and the seventh days were determined.
     Tissue extraction method for adenine nucleotides: After preservation, a part of pancreas was rapidly frozen in liquid nitrogen, lyophilized overnight, and kept at -80 °C until analysis. The dry tissue powder was weighed (200 mg) and homogenized in 3 mL of ice cold 0.5 mol/L perchloric acid. The precipitated protein was removed by centrifugation, and 500 mL of supernatant was neutralized by the additions of 50 mL 1 mol/L KOH and 50 mL Tris. Following centrifugation, 10 mL of supernatant was injected into HPLC for analysis.

Measurement of adenine nucleotides
High-performance liquid chromatography (HPLC, Waters, 510 Pump, 486 Detector) was performed on a reverse-phase column of Shim-pack, CLC-ODS (15 cm×3.96 mm, 4 mm) which was equilibrated with 100 mmol/L sodium phosphate buffer (pH 6.0) according to the method of Wynants et al.
    TAN was calculated as the sum of ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP).

Histological studies
Biopsies of the pancreas grafts were taken after cold preservation and one hour after transplantation. For light microscopy, the tissues were fixed in 100 ml/L formalin and stained with hematoxylin and eosin. For electron microscopic studies the tissues were prefixed in 25 g/L glutaraldehyde, postfixed in 25 g/L osmium tetroxide, sectioned at 1 mm, and stained with uranyl acetate and lead citrate.

Statistical analysis
All data were expressed as mean±SD. F test was used to compare values of different groups, x² test for comparison of viability. A value of P<0.05 was considered statistically significant.

RESULTS
Graft function
Pancreatic graft endocrine function  Plasma glucose and IVGTT K values in groups 1, 2, 3a and 3b recovered to normal 2-3 days after transplantation, while groups 3c and 3d did not one week after transplantation(Table1). The plasma insulin levels in splenic and peripheral veins in groups 1, 2, 3a, and 3b were much higher than those in groups 3c, and 3d (P<0.05, Table 2).
Pancreatic graft exocrine function  The pancreatic juice flow over the pancreatic duct 30 min after transplantation increased gradually and came to a climax on the fourth day after transplantation, and then declined gradually. The daily amounts of pancreatic juice of groups 1, 2, 3 a and 3b were much more than those of groups 3c and 3d (P<0.05). The amylase activities in the pancreatic juice of the first and seventh days in groups 1, 2, 3a and 3b  were much more than those in groups 3c and 3d (P<0.05, Tables 2, 3)
Tissue ATP, ADP, AMP and TAN after preservation  The tissue concentrations of ATP, ADP, AMP and TAN after 24-hour preservation in groups 1, 2, 3 a and 3b were much higher than those in groups 3c and 3d (P<0.05, Table 4).
Viability of canine pancreas autografts after preservation   After significant warm and cold preservation, the functional success rates of groups 2, 3a, 3b, 3c and 3d were 5/5(100%), 6/6(100%), 6/6(100%), 4/6(66.7%) and 0/4(0%), respectively(Table3). The UW cold preservation method was effective for functional recovery of the pancreas after 30 to 60-min warm ischemia (Table 3).
Relationship between posttransplantation viability and tissue ATP and TAN  There was no overlap between the lowest ATP in the viable grafts and highest ATP in the nonviable grafts. If ATP level of 4.0 mmol/g dry weight was determined as a critical value for the viability following transplantation, the specificity, sensitivity, predictive value and efficacy were all 100%. And there was also no overlap between the lowest TAN in the viable grafts and highest TAN in the nonviable grafts. If TAN level of 7.0 mmol/g dry weight was determined as a critical value for the viability following transplantation, specificity, sensitivity, predictive value and efficacy were all 100%. Both ATP and TAN were reliable markers for determining the transplantation.

Table 1  Plasma glucose and IVGTT K value at the first week after transplantation and plasma insulin level in splenic and peripheral vein one hour after transplantation (mean±SD) 

¹F=36.9, P<0.05; ²F=32.9, P<0.05; ³F=7.38, P<0.05; ⁴F=38.2, P<0.05; 5SNK test: between group 1, 2, 3a, 3b, P>0.05; between group 3c, 3d, P>0.05.

Table 2  Pancreatic juice flow during the first week after transplantation (mean±SD, mL)

¹F=9.87, P<0.05; ²F=25.9, P<0.05; ³F=67.4, P<0.05; ⁴F=88.2, P<0.05; ⁵F=45.2, P<0.05; ⁶F=15.9, P<0.01; ⁷F=13.2, P<0.01; 8SNK test: between group 2, 3a, 3b, P>0.05; between group 3c, 3d, P>0.05; 9SNK test: between group 2, 3a, 3b, P>0.05.

Table 3  Amylase in pancreatic juice of the first and the seventh day and viability after significant warm and cold preservation (mean±SD)

¹F=10.3, P<0.05; ²F=205.5, P<0.05; 3SNK test: between group 2, 3a, 3b, P>0.05; 4SNK test: between group 2, 3a, 3b, P>0.05; between group 3c, 3d, P>0.05. 5compare with Group 2, P<0.05.

Table 4  Tissue concentration of ATP, ADP, AMP and TAN (mean±SD,mmol/L) 

¹F=17.0, P<0.05; ²F=23.9, P<0.05; 3SNK test: between group 2, 3a, 3b, P>0.05.

Histologic studies
Under light microscope, the pancreas in groups 1 and 2 stored for 24 h showed normal architecture. After 24 h, preservation vacuolization of the acinar cells and interstitial edema were seen in grafts of groups 3a and 3b, and only mild edema of the islets was evident. Grafts of groups 3c and 3d showed severe edema, and after revascularization there was hemorrhage in the interstitial space.
       Under electron microscope, the pancreas in groups 1 and 2 stored for 24 h  showed well preserved cells. In grafts of groups 3a and 3b, acinar cells showed no nuclear changes, but rough endoplasmic reticulum (RER) cisternae were dilated. In grafts of groups 3c and 3d, irreversible cell damage was seen in most, but not all, specimens.

DISCUSSION
Pancreas graft injury due to warm ischemia strongly affects the posttransplant outcome^[16,17]. Therefore, resuscitation of an ischemically damaged pancreas is essential to enlarge the donor pool using the pancreas graft from the cardiac arrest donor^[18]. We have demonstrated that canine pancreases subjected to 60 min of warm ischemia can be resuscitated during preservation by the UW solution preservation method at 4 °C for 24 h.
     Restoration of cellular function of the pancreas graft subjected to significant warm ischemia by the UW solution cold preservation method will make it possible to use pancreas grafts from cadaver with cardiac arrest^[19,20], wait safely for the excision of the pancreas and enlarge the donor pool. Cerra^[21,22]reported that  the canine pancreatic allografts tolerated warm ischemia up to one hour. Florack et al.^[23,24]demonstrated that the canine pancreatic autografts tolerated warm ischemia up to 60 min.
     On the other hand, the assessment of a pancreas graft viability before transplantation is very important to prevent transplantation of a nonfunctioning allograft especially after significant warm ischemia because there is progressive depletion of ANs during warm ischemia^[25], ultimately leading to ischemic damage. But the relationship between the tissue concentration of ANs before transplantation and organ viability after transplantation is controversial^[26,27]. In human liver preservation, Lanir et al.^[28,29] demonstrated a direct correlation between a high ATP concentration and good posttransplant outcome. On the contrary, correlation between the ATP level at the end of cold preservation and viability following transplantation was not proved in the rat liver^[30,31].
      We have also demonstrated that correlation between high  ATP tissue concentration, which is necessary to maintain cellular integrity, and good posttransplant outcome of a canine pancreas graft after preservation by the UW solution cold preservation method^[32,33]. It is suggested that tissue concentration of ATP and TAN at the end of 24-h preservation by the UW solution cold preservation method will predict the posttransplant outcome of pancreas graft subjected to significant warm ischemia^[34,35]. But the mechanism responsible for the effectiveness of the UW solution cold preservation method in restoration of function of the pancreas graft subjected to significant warm ischemia remains unclear and is under investigation^[35-38].
     We conclude that the tissue concentration of ATP and TAN at the end of 24-h preservation by the UW solution cold storage method will predict the posttransplant outcome of pancreas graft subjected to significant warm ischemia.

REFERENCES
1    Pi F, Badosa F, Sola A, Rosello Catafau J, Xaus C, Prats N, Gelpi E, Hotter G. Effects of adenosine on
      ischaemia-reperfusion injury associated with rat pancreas transplantation. Br J Surg 2001; 88: 1366-1375
2    Uhlmann D, Armann B, Ludwig S, Escher E, Pietsch UC, Tannapfel A, Teupser D, Hauss J, Witzigmann H. Comparison of
      Celsior and UW solution in experimental pancreas preservation. J Surg Res 2002; 105: 173-180
3    Wakai A. Effect of adenosine on ischaemia-reperfusion injury associated with rat pancreas transplantation. Br J Surg
      2002; 89: 494
4    Thayer SP, Fernandez-del Castillo C, Balcom JH, Warshaw AL. Complete dorsal pancreatectomy with preservation of the
      ventral pancreas: a new surgical technique. Surgery 2002; 131: 577-580
5    Fujita H, Kuroda Y, Saitoh Y. The mechanism of action of the two-layer cold storage method in canine pancreas
      preservation-protection of pancreatic microvascular endothelium. Kobe J Med Sci 1995; 41: 47-61
6    Matsumoto S, Kandaswamy R, Sutherland DE, Hassoun AA, Hiraoka K, Sageshima J, Shibata S, Tanioka Y, Kuroda Y.
      Clinical application of the two-layer (University of Wisconsin solution/perfluorochemical plus O2) method of pancreas
      preservation before transplantation. Transplantation 2000; 70: 771-774
7    Sun B, Jiang HC, Piao DX, Qiao HQ, Zhang L. Effects of cold preservation and warm reperfusion on rat fatty liver. World J
      Gastroenterol 2000; 6: 271-274
8    Kuroda Y, Tanioka Y, Matsumoto S, Hiraoka K, Morita A, Fujino Y, Suzuki Y, Ku Y, Saitoh Y. Difference in energy
      metabolism between fresh and warm ischemic canine pancreases during preservation by the two-layer method. Transpl
      Int 1994; 7(Suppl 1): S441-445
9    Randhawa P. Allograft biopsies in management of pancreas transplant recipients. J Postgrad Med 2002; 48: 56-63
10  Kim Y, Kuroda Y, Tanioka Y, Matsumoto S, Fujita H, Sakai T, Hamano M, Suzuki Y, Ku Y, Saitoh Y. Recovery of pancreatic
      tissue perfusion and ATP tissue level after reperfusion in canine pancreas grafts preserved by the two-layer method.
      Pancreas 1997; 14: 285-289
11  Matsumoto S, Kuroda Y, Fujita H, Tanioka Y, Sakai T, Hamano M, Kim Y, Suzuki Y, Ku Y, Saitoh Y. Extending the margin 
      of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the
      two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis
      inhibitor OKY046. Transplantation 1996; 62: 879-883
12  Obermaier R, Benz S, Kortmann B, Benthues A, Ansorge N, Hopt UT. Ischemia/reperfusion-induced pancreatitis in rats: 
      a new model of complete normothermic in situ ischemia of a pancreatic tail-segment. Clin Exp Med 2001; 1: 51-59
13  Matsumoto S, Kuroda Y, Fujita H, Tanioka Y, Kim Y, Sakai T, Hamano M, Suzuki Y, Ku Y, Saitoh Y. Resuscitation of
      ischemically damaged pancreas by the two-layer (University of Wisconsin solution/perfluorochemical) mild hypothermic
      storage method. World J Surg 1996; 20: 1030-1034
14  Tanioka Y, Kuroda Y, Kim Y, Matsumoto S, Suzuki Y, Ku Y, Fujita H, Saitoh Y.The effect of ouabain (inhibitor of an
      ATP-dependent Na+/K+ pump) on the pancreas graft during preservation by the two-layer method. Transplantation 
      1996; 62: 1730-1734
15  Moorhouse JA, Granhame GR, Rosen NJ. Relationship between intravenous glucose tolerance and the fasting blood
      glucose level in healthy and diabetic subjects. J Clin Endocrinnol Metab 1964; 24: 145-159
16  Troisi R, Meester D, Regaert B, van den Broecke C, Cuvelier C, Hesse UJ. Tolerance of the porcine pancreas to warm 
      and cold ischemia: comparison between University of Wisconsin and histidine-tryptophan-ketoglutarate solution.
      Transplant Proc 2002; 34: 820-822
17  Fujino Y, Suzuki Y, Tsujimura T, Takahashi T, Tanioka Y, Tominaga M, Ku Y, Kuroda Y. Possible role of heat shock protein
      60 in reducing ischemic-reperfusion injury in canine pancreas grafts after preservation by the two-layer method. 
      Pancreas 2001; 23: 393-398
18  Ludwig S, Armann B, Escher E, Gabel G, Teupser D, Tannapfel A, Pietsch UC, Hauss J, Witzigmann H, Uhlmann D.
      Pathomorphologic and microcirculatory changes and endothelin-1 expression in UW-and Celsior-preserved pancreata 
      in experimental pancreas transplantation. Transplant Proc 2002; 34: 2364-2365
19  Lakey JR, Kneteman NM, Rajotte RV, Wu DC, Bigam D, Shapiro AM. Effect of core pancreas temperature during cadaveric
      procurement on human islet isolation and functional viability. Transplantation 2002; 73: 1106-1110
20  Nagami H, Tamura K, Kin S, Teramoto M, Ishida T. Beneficial effect of thromboxane A2 synthetase inhibitor (OKY-046) 
      on 24-hr simple hypothermic preservation of the canine pancreas graft. Int Surg 1995; 80: 274-277
21  Cerra FB, Adams JR, Eggert DE, Eilert JB, Bergan JJ. Pancreatic function after normothermic ischemia. II. Cadaveric
      transplanatation. Am J Surg 1970; 120: 693-696
22  Tsiotos GG, Sarr MG. Pancreas-preserving total duodenectomy. Dig Surg 1998; 15: 398-403
23  Florack G, Sutherland DE, Ascherl R, Heil J, Erhardt W, Najarian JS. Definition of normothermic ischemia limits for kidney
      and pancreas grafts. J Surg Res 1986; 40: 550-563
24  Tanioka Y, Sutherland DE, Kuroda Y, Suzuki Y, Matsumoto I, Deai T. Preservation of dog pancreas before islet isolation
      with the two-layer method. Transplant Proc 1998; 30: 3419-3420
25  Iwanaga Y, Suzuki Y, Okada Y, Mori H, Matsumoto I, Mitsutsuji M, Tanioka Y, Fujino Y, Tominaga M, Ku Y, Kuroda Y.
      Ultrastructural analyses of pancreatic grafts preserved by the two-layer cold-storage method and by simple cold storage
      in University of Wisconsin solution. Transpl Int 2002; 15: 425-430
26  Belzer FO, Southard JH. Principles of solid-organ preservation by cold storage. Transplantation 1988; 45: 673-676
27  Kim Y, Kuroda Y, Tanioka Y, Matsumoto S, Fujita H, Sakai T, Hamano M, Suzuki Y, Ku Y, Saitoh Y. Recovery of adenosine
      triphosphate tissue levels of grafts preserved by the two-layer method after reperfusion. Artif Organs 
      1996; 20: 1120-1124
28  Lanir A, Jenkins RL, Caldwell C, Lee RG, Khettry U, Clouse ME. Hepatic transplantation survival: correlation with adenine
      nucleotide level in donor liver. Hepatology 1988; 8: 471-475
29  Tsujimura T, Suzuki Y, Takahashi T, Yoshida I, Fujino Y, Tanioka Y, Li S, Ku Y, Kuroda Y. Successful 24-h preservation 
      of canine small bowel using the cavitary two-layer (University of Wisconsin solution/perfluorochemical) cold storage
      method. Am J Transplant 2002; 2: 420-424
30  Orii T, Ohkohchi N, Satomi S, Taguchi Y, Mori S, Miura I. Assessment of liver graft function after cold preservation using
      31P and 23Na magnetic resonance spectroscopy. Transplantation 1992; 53: 730-734
31  Uhlmann D, Armann B, Ludwig S, Escher E, Pietsch UC, Tannapfel A, Teupser D, Hauss J, Witzigmann H. Comparison of
      Celsior and UW solution in experimental pancreas preservation. J Surg Res 2002; 105: 173-180
32  Lakey JR, Rajotte RV, Warnock GL, Kneteman NM. Cold ischemic tolerance of human pancreas: assessment of islet
      recovery and in vitro function. Transplant Proc 1994; 26: 3416
33  Shi LJ, Liu JF, Zhang ZQ, Lu YQ, Shu YG, Chen GL, Xin ZH, Xu JY. Alterations of ATPase activity and erythrocyte oxygen
      consumption in patients with liver-blood deficiency syndrome. China Nati J New Gastroenterol 1997; 3: 180-181
34  Humar A, Kandaswamy R, Drangstveit MB, Parr E, Gruessner AG, Sutherland DE. Prolonged preservation increases
      surgical complications after pancreas transplants. Surgery 2000; 127: 545-551
35  Bottino R, Balamurugan AN, Bertera S, Pietropaolo M, Trucco M, Piganelli JD. Preservation of human islet cell functional
      mass by anti-oxidative action of a novel SOD mimic compound. Diabetes 2002; 51: 2561-2567
36  Kim Y, Kuroda Y, Tanioka Y, Matsumoto S, Fujita H, Sakai T, Hamano M, Suzuki Y, Ku Y, Saitoh Y. Recovery of pancreatic
      tissue perfusion and ATP tissue level after reperfusion in canine pancreas grafts preserved by the two-layer method.
      Pancreas 1997; 14: 285-289
37  Pi F, Hotter G, Closa D, Prats N, Fernandez-Cruz L, Badosa F, Gelpi E, Rosello-Catafau J. Differential effect of nitric oxide
      inhibition as a function of preservation period in pancreas transplantation. Dig Dis Sci 1997; 42: 962-971
38  Matsumoto I, Suzuki Y, Fujino Y, Tanioka Y, Deai T, Iwanaga Y, Mitsutsuji M, Iwasaki T, Tominaga M, Ku Y, Kuroda Y.
      Superiority of mild hypothermic (20 degrees C) preservation for pancreatic microvasculature using the two-layer 
      storage method. Pancreas 2000; 21: 305-309

   Edited by Hu DK and Zhu LH  Proofread by Xu FM  

Reviews	Add
more>>