JOURNAL
OF SURGICAL
RESEARCH
A New Technique
of Orthotopic en Bloc Transplantation Liver and Duct-Ligated Pancreas1
HINRICH BITTER-SUERMANN, HA;KAN GKBEL, *Department tDeparrment
27, 105- 113 (1979)
of the Porcine
M.D.,* HANS ~RYNGER, M.D. ,t INGEMAR WIKSTR~~M, M.D.,t JOHAN SAVE-WDERBERGH, M.D.,* AND LARS-ERIK GELIN, M.D.t
M.D.,t
of Pathology, School of Medicine, Georgetown University, Washington, D. C. 20007, and of Surgery 1, Sahlgrensko Sjukhuset, Universify of Gothenburg, 41345 Gothenburg, and ~Deparzment of Pathology bstra Sjukhusec, 41685 Gorhenburg, Sweden Received
August 15, 1977
A new technique of transplanting porcine liver and pancreas en bloc in an orthotopic position is described. The pancreatic duct was ligated. Recipients were made diabetic either by total pancreatectomy or by intravenous administration of streptozotocin, (100 m&g body wt). Careful correction of electrolyte and fluid disturbances in recipient animals was mandatory before and after surgery. No rejection of either liver or pancreas grafts was seen. The endocrine function of the pancreatic grafts was adequate.
Reports of pancreatic transplantation in pigs are rare. In 1972, Calne et al. and Sells ef al. described orthotopic pancreas allografts in pigs using part of the duodenum as a conduit for drainage of the pancreatic juice [6,22]. The Cambridge team had previously established reproducible survival of pig liver allografts in the absence of immunosuppression and had demonstrated the protective effect of such liver allografts on other organ grafts obtained from the liver donor [4,5]. En some experiments the liver was transplanted en bloc with the kidney and pancreas and seven out of nine pigs showed good endocrine pancreas function up to 57 days after transplantation. A duodenal conduit drained the pancreatic juice [6]. Two long-term surviving pigs with pancreatic allografts in the iliac fossa had an adequate graft function [26]. These pigs were immunosuppressed with azathioprine and the pancreatic juice was drained to the recipients’ small bowel via a duodenal conduit. Recently, Kyriakides et al. described 1 This study was supported by the Tore Nilson Foundation, Stockholm, Sweden, Grant No. 76-75, by the Swedish Medical Research Council, Grant No. B7517X549-lDB, and by a grant from the Wasa Foundation, Gothenburg, Sweden.
segmental duct-ligated porcine autografts and allografts with anastomoses to the vessels of the neck 113, 141. The issue of whether islets or wholeorgan pancreas grafts are preferable as possible future therapy in the treatment of diabetes is controversial [15]. The current increasing volume of information on islet graft-related topics contrasts with the scanty data from the few experiments involving whole-organ pancreas grafts. Isolated islet isografts permanently cured rats of diabetes Cl, 121, whereas isolated islet allografts in mice, rats, and man seemed to be extremely susceptible to rejection phenomena [S, 9, 16-18, 20, 251. On the other hand, duct-ligated pancreas allografts in DL-A matched beagles fared well for long periods of time [71, as did duct-ligated isografts in rats [3, 19, 211. We have shown, in an earlier report, that duct-ligated pancreas allografts in diabetic rats made unresponsive by spleen allografts survived indefinitely 131. The aim of this investigation was to induce diabetes in pigs, either by total pancreatectomy or by the injection of streptozotocin, and then to transplant such animals en bloc with liver and duct-ligated pan-
105
0022-4804/79/080105-09$01.00/0 CDpyri$ht 0 1979 by Academic Press,Inc. AU rishls
of reproduction
in any form reserved
106
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 27, NO. 2, AUGUST
1979
creatic grafts. Functional parameters of the Experimental Groups pancreatic grafts could therefore be studied Group I. Five pigs underwent total panunder the immunologically privileged condi- createctomy. tions created by the liver transplant. Group 2. Five pigs were rendered diabetic with an intravenous injection of strepMATERIALS AND METHODS tozotocin (100 mg/kg body wt) into a jugular vein. Animals Group 3. Six animals received a convenDonors were Swedish white Landrace tional orthotopic liver allograft. pigs of either sex and 20-25 kg body wt. Group 4. Six animals underwent total Similar English white Landrace pigs served pancreatectomy and, immediately thereas recipients. The animals were starved 24 after, were transplanted with an orthotopic hr prior to surgery. Postoperatively, the pigs liver-pancreas graft. were kept in a warm environment and, 24 hr Group 5. Nine animals, rendered diabetic after surgery, were allowed food ad libitum. with streptozotocin 5- 11 days prior to surgery, were transplanted with an orthoAnesthesia topic liver-pancreas graft. The animals were premeditated with OPERATIVE TECHNIQUES atropine and anesthesia was induced with halothane and oxygen. The trachea was intubated and artificial ventilation was per- Donor formed using a 1:1 mixture of nitrous oxide The objective of the donor operation was and oxygen together with halothane (O.l- to procure a liver and a pancreas with an 0.8%). The mean arterial pressure and cen- undisturbed vascular attachment of the pan: tral venous pressure were monitored in all cress to the celiac artery and the portal vein. recipient animals. Lost blood was replaced Thus, both organs would have the same with donor blood and a 4: 1 mixture of 5 5% blood supply and drainage after transplantaglucose and 10% human albumin. Post- tion. Through a midline incision, the panoperatively, the animals received an intra- creas was carefully dissected from the survenous infusion containing 10% glucose, rounding structures with division of all human albumin, sodium bicarbonate, and ligamentous attachments. The pancreatic potassium if necessary. duct was ligated and divided near the duodenum. Thereafter, all arteries arising from Laboratory Monitoring the celiac artery, except for the hepatic During the recipient operation, blood artery, were ligated and divided away from samples were taken every 15 min for analy- the pancreas so as to leave its arterial sis of blood gases, acid-base balance, elec- blood supply undisturbed. The liver was trolytes, hemoglobin, and glucose levels. then skeletonized, providing a good view Such sampling was mandatory immediately of the suprahepatic inferior vena cava after revascularization of the donor liver- (IVC), the infrahepatic IVC, and the compancreas specimen. After surgery, pan- mon bile duct which was divided. On comcreatic graft function was assessed by pletion of the liver dissection, the animals determination of blood glucose levels during were exsanguinated via the aorta. The blood fasting and after an intravenous injection of was collected and used for transfusion into a 50% solution of glucose (1 g/kg body wt). the recipients. The liver was perfused via The glucose tolerance was expressed as a the superior mesenteric vein with 2 liters of Ringer-lactate solution at 4”C, to which K value [ll].
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
107
1 g of ampicillin and 100 mg of methylprednisolone were added. The liver was then removed, taking great care to ligate the three diaphragmatic veins joining the suprahepatic IVC. A Carrel patch of the aorta was left at the celiac artery and the superior mesenteric vein was cut 0.5 cm distal to the pancreas. After removal of the liver-pancreas specimen, 500 ml of the perfusate was flushed through the celiac artery to provide a washout of the pancreas. The specimen was then stored at 4°C until transplantation with a mean cold ischemic time, until revascularization, of 3 hr. Recipient
In group 4 animals, as in the group 1 controls, the whole pancreas was dissected from the surrounding structures and all vessels coming from the splenic and pancreaticoduodenal arteries were identified and ligated. The pancreas was removed without interfering with the blood supply of either the spleen or the duodenum. In group 5, no pancreatectomy was performed, as the animals were already streptozotocindiabetic. Following total pancreatectomy in group 4, the operative procedure in both groups 4 and 5 was identical to conventional orthotopic liver transplantation in the pig, based on the technique of Caine et al. [4]. The recipient liver was removed and in the unhepatic phase portal blood was shunted to the left external jugular vein. The liverpancreas specimen was then transplanted using running over-and-over sutures. The sequence of anastomoses was as follows (see Fig. 1): 1. Suprahepatic IVC to suprahepatic WC, end-to-end. 2. Donor superior mesenteric vein to the recipient portal vein, end-to-end. At this stage, the clamps at the suprahepatic IVC and portal vein were released and the liver was linked to the portal circulation. 3. The aorta was cross-clamped. The celiac artery with a Carrel patch of the aorta
FIG. 1. Diagram of orthotopic liver-pancreas transplantation without drainage of pancreatic juice. Pancreatic vascular connections to celiac artery and portal vein are undisturbed. Anastomoses are accomplished in the following order: (1) suprahepatic IVC to suprahepatic IVC, end-to-end; (2) superior mesenteric vein to portal vein, end-to-end; (3) celiac artery with Carrel patch to aorta, end-to-side; (4) infrahepatic IVC to infrahepatic IVC, end-to-end; (5) common bile duct to common bile duct, end-to-end without T-tube. Technique identical for group 4 animals (with preceding total pancreatectomy for induction of diabetes) and group 5 animals (without preceding pancreatectomy, since they were streptozotocin-diabetic).
was joined end-to-side to the recipient aorta, near the superior mesenteric artery. After release of the aortic clamps, the pancreas received its first blood supply. 4. The infrahepatic IVC was anastomosed end-to-end to the recipient IVC. 5. Finally, the common bile ducts were joined end-to-end. In conventional orthotopic pig liver transplantation, donor and recipient portal veins are anastomosed end-to-end, after trimming of the donor vessel to prevent kinking of the anastomosis. In our experiments, no trimming of the donor portal vein was possible because the pancreas drains into this ves-
108
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979 TABLE 1 GROUP 1: SURVIVAL FOLLOWING TOTAL PANCREATECTOMY
Survival @w) 1 2 3 4 5
Cause of death
7 7 8 8 10
Emaciation Emaciation Emaciation Emaciation Emaciation
and and and and and
diabetes diabetes diabetes diabetes diabetes
sel. Thus, the donor superior mesenteric vein with the attached pancreas had to be anastomosed end-to-end to the recipient portal vein. It was necessary to fix the pancreas to the surrounding structures at two points in order to avoid kinking of this anastomosis. The abdomen was drained with silastic tubes and the wound closed with a running over-and-over suture of nylon for the muscle and chromic catgut for the skin. A catheter in the left external jugular vein was guided subcutaneously to the back of the neck and left in situ throughout the lifetime of the animals. RESULTS The results are summarized in Tables 1-5. The pancreatectomized animals in group 1 had a severely disturbed glucose metabo-
lism, with their general condition rapidly deteriorating. They all died within 10 days. All animals in group 2 became diabetic with a K value of 0.6 5 0.2 (SD) 5-l 1 days after streptozotocin injection, compared to a K value of 2.6 + 0.6 (SD), (P < O.OOl), before the injection. Following successful induction of diabetes, the animals were treated with insulin. K values were determined after periodic withdrawal of insulin. No animal showed a recovery of islet function within an observation time of up to 7 months. On histological examination, the islet structure was dissolved and no p-cells could be seen. Group 3 animals receiving conventional orthotopic liver allografts showed no signs of rejection. Two long-term surviving pigs were sacrificed in good health. Three animals in group 4 lived for 5, 6, and 28 days, respectively, following orthotopic liver-pancreas allografts after total pancreatectomy. The cause of death was not related to failure of either liver or pancreas grafts. The latter had a normal appearance at postmortem and histological evaluation demonstrated healthy islets. In all three animals, fasting blood glucose and K values were adequate, except in pig no. 6 which suffered from deteriorating endocrine graft function 3 weeks after transplantation possibly related to repeated gastric hemorTABLE 3 GROUP 3: RESULTS OF ORTHOTOPIC LIVER TRANSPLANTATION
TABLE 2 GROUP 2: EFFECT OF STREPTOZOTOCIN INJECTION (100 mg/kg body wt) ON GLUCOSE TOLERANCE K Values 5-11
streptozotocin injection
days after streptozotocin injection
2.7 2.0 3.5 2.1 2.1
0.4 0.5 0.8 0.7 0.6
K Values before
a 1 2 3 4 5
fig
Survival (days)
1 2
6 18
3 4 5 6
83 134 150 244
Cause of death Gastric hemorrhage Biliary fistula and peritonitis Intestinal obstruction Intestinal obstruction Sacrificed in good health Sacrificed in good health
Grab function until death + + + + + +
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
109
TABLE 4 GROUP 4: RESULTS OF ORTHOTOPIC LIVER AND PANCREAS TRANSPLANTATION, DIABETES INDUCED BY TOTAL PANCREATECTOMY
m
Survival
Cause of death
Comments
1
0
Cardiac arrest on perfusion of liver with portal blood
2
0
Cardiac arrest on perfusion of liver with portal blood
3
8 hr
Hemorrhage
4
5 Days
Gastric hemorrhage
Excellent function of liver-pancreas graft until death
5
6 Days
Respiratory insufficiency due to bilateral hydrothorax
Excellent function of liver-pancreas graft until death
6
28 Days
Gastric hemorrhage
Until 3 weeks after transplantation, excellent function of liver-pancreas graft which deteriorated in connection with repeated gastric hemorrhages
TABLE 5 GROUP 5: RESULTS OF ORTHOTOPIC LIVER AND PANCREAS TRANSPLANTATION, DIABETES INDUCED BY STREPTOZOTOCIN
Survival
Cause of death
Comments
1
0
Cardiac arrest on perfusion of liver with portal blood
2
1 hr
Septicemia and hyperpyrexia resulting in circulatory insufficiency
Diabetes induced 11 days prior to transplantation; known wound infection from jugular catheter
2 hr
Cardiac arrest
Grave ketosis at start of surgery
2 hr
Cardiac arrest
Grave ketosis at start of surgery
3 hr
Hemorrhage due to poor liver function
Cardiac arrest during surgery resulting in prolonged hypotension with bad perfusion of liver graft
6
5 Days
Intestinal obstruction
Excellent function of liver-pancreas graft until death
7
6 Days
Respiratory insufficiency due to bilateral hydrothorax
Excellent function of liver-pancreas graft until death
8
6 Days
Small bowel perforation and peritonitis
Excellent function of liver-pancreas graft until death
9
22 Days
Intestinal obstruction
Excellent function of liver-pancreas graft until death
110
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979
160 2 E E 120
I
:it : 2 ;L 2
----, 00
,/-I I
e-
,’ /’ /’
I
3
40
4
-1 : z
2
h, I 10
20
I 30
40
I 30
1 60
I 70
MlllUl~,
FIG. 2. Changes in mean arterial pressure of 12 animals ingroups 4 and 5 during recipient operation, (mean + SD). (1) removal of liver after clamping of IVC and establishing a portojugular shunt; (2) liver perfused with portal blood; (3) cross-clamping of aorta for arterial anastomosis; (4) clamps off aorta; (5) clamps off infrahepatic IVC.
rhages. Two animals died intraoperatively after the anastomoses of the suprahepatic IVC and the portal vein had been accomplished and portal blood was allowed to perfuse the liver. Both animals had a cardiac arrest. It was obvious that the animals were generally in poor condition following the time-consuming pancreatectomy that preceded the liver-pancreas grafting, and their blood pressure was often low after prolonged handling of the gut. This was an undesirable and sometimes fatal feature, since the blood pressure fell again in the unhepatic phase (see Fig. 2) and the liver was finally insufficiently perfused with blood containing accumulated toxic metabolites from the gut. In group 5 recipients with streptozotocininduced diabetes, four out of nine animals survived transplantation between 5 and 22 days and had excellent liver-pancreas function throughout their lifetime. At postmortem, the pancreatic grafts had a normal appearance without surrounding fluid collection, edema, or fibrosis. The p-cells had an almost normal histological appearance (see Figs. 3 and 4). Four pigs in this group died in the early postoperative period, which reflects their poor health due to the untreated diabetes induced between 5 and
11 days preoperatively. Once the potential diabetic recipients were subjected to a strict preoperative treatment, with a correction of the prevailing ketosis and fluid and electrolyte disorders, early postoperative deaths no longer occurred. While animals in group 4 recovered slowly from the side effects of total pancreatectomy and seldom started to feed during the first 2-3 days postoperatively, group 5 recipients were usually alert and in good condition on the day after surgery. Surviving pigs in groups 4 and 5 had a conspicuous consumption of glucose and potassium immediately after transplantation which had to be replaced accordingly. DISCUSSION This study shows that the phenomenon of survival of non-duct-ligated porcine pancreas allografts, in the presence of orthotopic liver allografts as described by Calne et al. [6, 221, can also be extended to ductligated pancreatic grafts. The mechanisms underlying the survival of pig liver allografts [4,24] and the donor-specific tolerogenic effect they exert in their new hosts are still obscure. The described technique of transplanting liver and duct-ligated pancreas en bloc in an orthotopic position is original and simple and resembles, in part, conventional liver transplantation in pigs 14, 231. In group 4 recipient animals, the blood pressure was already low after the timeconsuming pancreatectomy preceding transplantation and it fell further in the unhepatic phase leading to undesirable effects such as an insufficiently perfused liver graft and an accumulation in the blood of toxic metabolites from the gut. This led us to change from total pancreatectomy to the intravenous injection of streptozotocin for the induction of diabetes, a procedure which had several advantages. First, the recipient operation was much shorter and the animals were in better condition, presenting a higher blood pressure in the very critical period of initial perfusion with
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
111
FIG. 3. Islet from normal pig pancreas. Gomori, x375.
portal blood of the newly transplanted liver. Second, the duodenum was not skeletonized and surviving pigs could, therefore, feed the day after surgery, whereas recipient animals in group 4 with total pancreatectomy usually took 2-3 days to recover,
during which period they had to be provided with intravenous alimentation. Furthermore, the presence of the hosts’ own streptozotocin-damaged pancreas, which retained normal exocrine function, was advantageous in this model designed for a
FIG. 4. Islet from transplanted pancreas, pig No. 7 in group 5. Almost normal appearance of islet cells. Gomori, x375.
112
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979
long-term study of the grafts, since the animals needed no exocrine substitution. Howpigs were ever, streptozotocin-diabetic high-risk recipients and survived the lengthy transplant operation only after the adoption of a thorough preoperative assessment and regulation of their diabetic disorders. This factor was not realized during the first five experiments in group 5 and a series of inexplicable cardiac arrests ensued, which were absent in the latter part of the series. This study comprises too few experiments to be conclusive. However, rejection of pancreas grafts did not occur in the presence of liver allografts during the lifetime of the animals. We shall pursue this study in order to obtain long-term surviving pigs. Such animals could be useful for an analysis of endocrine pancreatic graft function in the absence of immunosuppression. ACKNOWLEDGMENT The authors thank Mrs. M. Oeltzschner, Anatomy Department, McGill University, Montreal, Canada, for the diagram.
REFERENCES 1. Ballinger, W. F., and Lacy, P. E. Transplantation of intact pancreatic islets in rats. Surgery 12: 175, 1972. 2. Bergan, J. J. ACSINIH Organ Transplant Reg. Newslett. 16, April 1976. 3. Bitter-Suermann, H., and Save-Soderbergh, J. The course of pancreas allografts in rats conditioned by spleen allografts. Transplantation 26: 28, 1978. 4. Calne, R. Y., White, H. J. O., Yoffa, D. E., Maginn, R. R., Binns, R. M., Samuel, J. R., and Molina, V. P. Observations of orthotopic liver transplantation in the pig. &if. Med. 1. 2: 478, 1%7. 5. Calne, R. Y., Sells, R. A., Pena, J. R., Davis, D. R., Millard, P. R., Herbertson, B. M., Binns, R. M., and Davies, D. A. L. Induction of immunological tolerance by porcine liver allografts. Nature (London) 223: 472, 1969. 6. Calne, R. Y., Sells, R. A., Marshall, V. C., Millard, P. R., Herbertson, B. M., Hadjiyannakis, E. J., Dunn, D. C., Robson, A. J., and Davis, D. R. Multiple organ grafts in the pig. Techniques and results of pancreatic, hepatic, cardiac and renal allografts. Brir. J. Surg. 59: 969, 1972.
7. de Cruyl, J., Westbroek, D. L., Dijkhuis, C. M., Vriesendorp, H. M., MacDicken, I., Elion-Gerritsen, W., Verschoor, L., Hulsmans, H. A. M., and Horchner, P. Influence of DL-A matching, ALS and 24-hour preservation on isolated pancreas allograft survival. Transplant. Proc. 5: 755, 1973. 8. Delmonico, F. L., Chase, C. M., and Russell, P. S. Transplantation of rat islets of Langerhans into diabetic mice. Transplant. Proc. 9: 367, 1977. 9. Frangipane, L. G., Poole, T. W., Barker, C. F., and Silvers, W. K. Vulnerability of allogeneic and xenogeneic pancreatic islets to alloantisera. Transplant. Proc. 9: 371, 1977.
lo. Gabel, H., Brynger, H., Bitter-Suermann, H., and
Henriksson, C. Surgically and chemically induced diabetes in pigs. Eur. Surg. Res. (Suppl. 1) 9: 285, 1977. 11. Hamilton, B., and Stein, A. F. The measurement of intravenous blood sugar curves. J. Lab. Clin. Med. 27: 491, 1942. 12 Kemp, C. B., Knight, M. J., Sharp, D. W., Bal’ linger, W. F., and Lacy, P. E. Effect oftransplantation site on results of pancreatic islet isografts in diabetic rats. Diabefologia 9: 486, 1973. 13. Kyriakides, G. K., Arora, V. K., Lifton, J., Nuttall, F. Q., and Miller, J. Porcine pancreatic transplantation. I. Autotransplantation of duct ligated segments. J. Surg. Res. 20: 451, 1976. 14. Kyriakides, G. K., Arora, V. K., Lifton, J., Nuttall, F. Q., and Miller, J. Porcine pancreatic transplants. II. Allotransplantation of duct ligated segments. J. Surg. Res. 20: 461, 1976. 15. Matas, A. J., Sutherland, D. E. R., and Najarian, J. S. Current status of islet and pancreas transplantation in diabetes. Diabetes 25: 785, 1976. 16. Najarian, J. S., Sutherland, D. E. R., Matas, A. J., Steffes, M. W., Simmons, R. L., and Goetz, F. C. Human islet transplantation: A preliminary report. Transplant.
Proc. 9: 233, 1977.
17. Nash, J. R., Peters, M., and Bell, P. R. F. Studies on the enhancement of rat islet allografts. Transplantation 25: 180, 1978. 18. Nelken, D., Friedman, E. A., Morse, S. I., and Beyer, M. M. Islet of Langerhans allotransplantation in the rat. Transplant. Proc. 9: 333, 1977. 19. Orloff, M. J., Lee, S., Charters, A. C., Grambort, D. E., Storck, L. G., and Knox, D. Long term studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg. 182: 198, 1975. 20. Reckard, C. R., and Barker, C. F. Transplantation of isolated pancreatic islets across strong and weak histocompatibility barriers. Transplant. Proc. 5: 761, 1973. 21. Reemtsma, K., Giraldo, N., Depp, D. A., and Eichwald, E. J. Islet cell transplantation. Ann. Surg. 168: 436, 1968. 22. Sells, R. A., Calne, R. Y., Hadjiyannakis, E. J.,
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
113
and Marshall, V. C. Glucose and insulin metabolism after pancreatic transplantation. Brir. Med. J.
survival of vascularized organ allografts in unimmunosuppressed pigs. Transplant. Proc. 5: 749,
3: 678, 1972. 23. Terblanche, J., Peacock, J. H., Bowes, J., and
1973. 25. Ziegler, M. M., Reckard, C. R., and Barker, C. F.
Hobbs, K. E. F. The technique of orthotopic liver homotransplantation in the pig. J. Surg. Res. 8: 151, 1968. 24. Terblanche, J., Hickman, R., Shippel, R. M., Dent, D. M., Spilg, H., Immelman, E. J., Du Toit, E., Uys, C. J., and Saunders, S. J. Prolonged
Long-term metabolic and immunological consideration in transplantation of pancreatic islets. J. Surg. Res. 16: 575, 1974. 26. Zimmermamt, G., Boeckl, O., Hell, E., and Seidl, P. Die iliakale Duodenopankreas Allotransplantation am Schwein. Chirurg 43: 372, 1972.
OF SURGICAL
RESEARCH
A New Technique
of Orthotopic en Bloc Transplantation Liver and Duct-Ligated Pancreas1
HINRICH BITTER-SUERMANN, HA;KAN GKBEL, *Department tDeparrment
27, 105- 113 (1979)
of the Porcine
M.D.,* HANS ~RYNGER, M.D. ,t INGEMAR WIKSTR~~M, M.D.,t JOHAN SAVE-WDERBERGH, M.D.,* AND LARS-ERIK GELIN, M.D.t
M.D.,t
of Pathology, School of Medicine, Georgetown University, Washington, D. C. 20007, and of Surgery 1, Sahlgrensko Sjukhuset, Universify of Gothenburg, 41345 Gothenburg, and ~Deparzment of Pathology bstra Sjukhusec, 41685 Gorhenburg, Sweden Received
August 15, 1977
A new technique of transplanting porcine liver and pancreas en bloc in an orthotopic position is described. The pancreatic duct was ligated. Recipients were made diabetic either by total pancreatectomy or by intravenous administration of streptozotocin, (100 m&g body wt). Careful correction of electrolyte and fluid disturbances in recipient animals was mandatory before and after surgery. No rejection of either liver or pancreas grafts was seen. The endocrine function of the pancreatic grafts was adequate.
Reports of pancreatic transplantation in pigs are rare. In 1972, Calne et al. and Sells ef al. described orthotopic pancreas allografts in pigs using part of the duodenum as a conduit for drainage of the pancreatic juice [6,22]. The Cambridge team had previously established reproducible survival of pig liver allografts in the absence of immunosuppression and had demonstrated the protective effect of such liver allografts on other organ grafts obtained from the liver donor [4,5]. En some experiments the liver was transplanted en bloc with the kidney and pancreas and seven out of nine pigs showed good endocrine pancreas function up to 57 days after transplantation. A duodenal conduit drained the pancreatic juice [6]. Two long-term surviving pigs with pancreatic allografts in the iliac fossa had an adequate graft function [26]. These pigs were immunosuppressed with azathioprine and the pancreatic juice was drained to the recipients’ small bowel via a duodenal conduit. Recently, Kyriakides et al. described 1 This study was supported by the Tore Nilson Foundation, Stockholm, Sweden, Grant No. 76-75, by the Swedish Medical Research Council, Grant No. B7517X549-lDB, and by a grant from the Wasa Foundation, Gothenburg, Sweden.
segmental duct-ligated porcine autografts and allografts with anastomoses to the vessels of the neck 113, 141. The issue of whether islets or wholeorgan pancreas grafts are preferable as possible future therapy in the treatment of diabetes is controversial [15]. The current increasing volume of information on islet graft-related topics contrasts with the scanty data from the few experiments involving whole-organ pancreas grafts. Isolated islet isografts permanently cured rats of diabetes Cl, 121, whereas isolated islet allografts in mice, rats, and man seemed to be extremely susceptible to rejection phenomena [S, 9, 16-18, 20, 251. On the other hand, duct-ligated pancreas allografts in DL-A matched beagles fared well for long periods of time [71, as did duct-ligated isografts in rats [3, 19, 211. We have shown, in an earlier report, that duct-ligated pancreas allografts in diabetic rats made unresponsive by spleen allografts survived indefinitely 131. The aim of this investigation was to induce diabetes in pigs, either by total pancreatectomy or by the injection of streptozotocin, and then to transplant such animals en bloc with liver and duct-ligated pan-
105
0022-4804/79/080105-09$01.00/0 CDpyri$ht 0 1979 by Academic Press,Inc. AU rishls
of reproduction
in any form reserved
106
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 27, NO. 2, AUGUST
1979
creatic grafts. Functional parameters of the Experimental Groups pancreatic grafts could therefore be studied Group I. Five pigs underwent total panunder the immunologically privileged condi- createctomy. tions created by the liver transplant. Group 2. Five pigs were rendered diabetic with an intravenous injection of strepMATERIALS AND METHODS tozotocin (100 mg/kg body wt) into a jugular vein. Animals Group 3. Six animals received a convenDonors were Swedish white Landrace tional orthotopic liver allograft. pigs of either sex and 20-25 kg body wt. Group 4. Six animals underwent total Similar English white Landrace pigs served pancreatectomy and, immediately thereas recipients. The animals were starved 24 after, were transplanted with an orthotopic hr prior to surgery. Postoperatively, the pigs liver-pancreas graft. were kept in a warm environment and, 24 hr Group 5. Nine animals, rendered diabetic after surgery, were allowed food ad libitum. with streptozotocin 5- 11 days prior to surgery, were transplanted with an orthoAnesthesia topic liver-pancreas graft. The animals were premeditated with OPERATIVE TECHNIQUES atropine and anesthesia was induced with halothane and oxygen. The trachea was intubated and artificial ventilation was per- Donor formed using a 1:1 mixture of nitrous oxide The objective of the donor operation was and oxygen together with halothane (O.l- to procure a liver and a pancreas with an 0.8%). The mean arterial pressure and cen- undisturbed vascular attachment of the pan: tral venous pressure were monitored in all cress to the celiac artery and the portal vein. recipient animals. Lost blood was replaced Thus, both organs would have the same with donor blood and a 4: 1 mixture of 5 5% blood supply and drainage after transplantaglucose and 10% human albumin. Post- tion. Through a midline incision, the panoperatively, the animals received an intra- creas was carefully dissected from the survenous infusion containing 10% glucose, rounding structures with division of all human albumin, sodium bicarbonate, and ligamentous attachments. The pancreatic potassium if necessary. duct was ligated and divided near the duodenum. Thereafter, all arteries arising from Laboratory Monitoring the celiac artery, except for the hepatic During the recipient operation, blood artery, were ligated and divided away from samples were taken every 15 min for analy- the pancreas so as to leave its arterial sis of blood gases, acid-base balance, elec- blood supply undisturbed. The liver was trolytes, hemoglobin, and glucose levels. then skeletonized, providing a good view Such sampling was mandatory immediately of the suprahepatic inferior vena cava after revascularization of the donor liver- (IVC), the infrahepatic IVC, and the compancreas specimen. After surgery, pan- mon bile duct which was divided. On comcreatic graft function was assessed by pletion of the liver dissection, the animals determination of blood glucose levels during were exsanguinated via the aorta. The blood fasting and after an intravenous injection of was collected and used for transfusion into a 50% solution of glucose (1 g/kg body wt). the recipients. The liver was perfused via The glucose tolerance was expressed as a the superior mesenteric vein with 2 liters of Ringer-lactate solution at 4”C, to which K value [ll].
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
107
1 g of ampicillin and 100 mg of methylprednisolone were added. The liver was then removed, taking great care to ligate the three diaphragmatic veins joining the suprahepatic IVC. A Carrel patch of the aorta was left at the celiac artery and the superior mesenteric vein was cut 0.5 cm distal to the pancreas. After removal of the liver-pancreas specimen, 500 ml of the perfusate was flushed through the celiac artery to provide a washout of the pancreas. The specimen was then stored at 4°C until transplantation with a mean cold ischemic time, until revascularization, of 3 hr. Recipient
In group 4 animals, as in the group 1 controls, the whole pancreas was dissected from the surrounding structures and all vessels coming from the splenic and pancreaticoduodenal arteries were identified and ligated. The pancreas was removed without interfering with the blood supply of either the spleen or the duodenum. In group 5, no pancreatectomy was performed, as the animals were already streptozotocindiabetic. Following total pancreatectomy in group 4, the operative procedure in both groups 4 and 5 was identical to conventional orthotopic liver transplantation in the pig, based on the technique of Caine et al. [4]. The recipient liver was removed and in the unhepatic phase portal blood was shunted to the left external jugular vein. The liverpancreas specimen was then transplanted using running over-and-over sutures. The sequence of anastomoses was as follows (see Fig. 1): 1. Suprahepatic IVC to suprahepatic WC, end-to-end. 2. Donor superior mesenteric vein to the recipient portal vein, end-to-end. At this stage, the clamps at the suprahepatic IVC and portal vein were released and the liver was linked to the portal circulation. 3. The aorta was cross-clamped. The celiac artery with a Carrel patch of the aorta
FIG. 1. Diagram of orthotopic liver-pancreas transplantation without drainage of pancreatic juice. Pancreatic vascular connections to celiac artery and portal vein are undisturbed. Anastomoses are accomplished in the following order: (1) suprahepatic IVC to suprahepatic IVC, end-to-end; (2) superior mesenteric vein to portal vein, end-to-end; (3) celiac artery with Carrel patch to aorta, end-to-side; (4) infrahepatic IVC to infrahepatic IVC, end-to-end; (5) common bile duct to common bile duct, end-to-end without T-tube. Technique identical for group 4 animals (with preceding total pancreatectomy for induction of diabetes) and group 5 animals (without preceding pancreatectomy, since they were streptozotocin-diabetic).
was joined end-to-side to the recipient aorta, near the superior mesenteric artery. After release of the aortic clamps, the pancreas received its first blood supply. 4. The infrahepatic IVC was anastomosed end-to-end to the recipient IVC. 5. Finally, the common bile ducts were joined end-to-end. In conventional orthotopic pig liver transplantation, donor and recipient portal veins are anastomosed end-to-end, after trimming of the donor vessel to prevent kinking of the anastomosis. In our experiments, no trimming of the donor portal vein was possible because the pancreas drains into this ves-
108
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979 TABLE 1 GROUP 1: SURVIVAL FOLLOWING TOTAL PANCREATECTOMY
Survival @w) 1 2 3 4 5
Cause of death
7 7 8 8 10
Emaciation Emaciation Emaciation Emaciation Emaciation
and and and and and
diabetes diabetes diabetes diabetes diabetes
sel. Thus, the donor superior mesenteric vein with the attached pancreas had to be anastomosed end-to-end to the recipient portal vein. It was necessary to fix the pancreas to the surrounding structures at two points in order to avoid kinking of this anastomosis. The abdomen was drained with silastic tubes and the wound closed with a running over-and-over suture of nylon for the muscle and chromic catgut for the skin. A catheter in the left external jugular vein was guided subcutaneously to the back of the neck and left in situ throughout the lifetime of the animals. RESULTS The results are summarized in Tables 1-5. The pancreatectomized animals in group 1 had a severely disturbed glucose metabo-
lism, with their general condition rapidly deteriorating. They all died within 10 days. All animals in group 2 became diabetic with a K value of 0.6 5 0.2 (SD) 5-l 1 days after streptozotocin injection, compared to a K value of 2.6 + 0.6 (SD), (P < O.OOl), before the injection. Following successful induction of diabetes, the animals were treated with insulin. K values were determined after periodic withdrawal of insulin. No animal showed a recovery of islet function within an observation time of up to 7 months. On histological examination, the islet structure was dissolved and no p-cells could be seen. Group 3 animals receiving conventional orthotopic liver allografts showed no signs of rejection. Two long-term surviving pigs were sacrificed in good health. Three animals in group 4 lived for 5, 6, and 28 days, respectively, following orthotopic liver-pancreas allografts after total pancreatectomy. The cause of death was not related to failure of either liver or pancreas grafts. The latter had a normal appearance at postmortem and histological evaluation demonstrated healthy islets. In all three animals, fasting blood glucose and K values were adequate, except in pig no. 6 which suffered from deteriorating endocrine graft function 3 weeks after transplantation possibly related to repeated gastric hemorTABLE 3 GROUP 3: RESULTS OF ORTHOTOPIC LIVER TRANSPLANTATION
TABLE 2 GROUP 2: EFFECT OF STREPTOZOTOCIN INJECTION (100 mg/kg body wt) ON GLUCOSE TOLERANCE K Values 5-11
streptozotocin injection
days after streptozotocin injection
2.7 2.0 3.5 2.1 2.1
0.4 0.5 0.8 0.7 0.6
K Values before
a 1 2 3 4 5
fig
Survival (days)
1 2
6 18
3 4 5 6
83 134 150 244
Cause of death Gastric hemorrhage Biliary fistula and peritonitis Intestinal obstruction Intestinal obstruction Sacrificed in good health Sacrificed in good health
Grab function until death + + + + + +
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
109
TABLE 4 GROUP 4: RESULTS OF ORTHOTOPIC LIVER AND PANCREAS TRANSPLANTATION, DIABETES INDUCED BY TOTAL PANCREATECTOMY
m
Survival
Cause of death
Comments
1
0
Cardiac arrest on perfusion of liver with portal blood
2
0
Cardiac arrest on perfusion of liver with portal blood
3
8 hr
Hemorrhage
4
5 Days
Gastric hemorrhage
Excellent function of liver-pancreas graft until death
5
6 Days
Respiratory insufficiency due to bilateral hydrothorax
Excellent function of liver-pancreas graft until death
6
28 Days
Gastric hemorrhage
Until 3 weeks after transplantation, excellent function of liver-pancreas graft which deteriorated in connection with repeated gastric hemorrhages
TABLE 5 GROUP 5: RESULTS OF ORTHOTOPIC LIVER AND PANCREAS TRANSPLANTATION, DIABETES INDUCED BY STREPTOZOTOCIN
Survival
Cause of death
Comments
1
0
Cardiac arrest on perfusion of liver with portal blood
2
1 hr
Septicemia and hyperpyrexia resulting in circulatory insufficiency
Diabetes induced 11 days prior to transplantation; known wound infection from jugular catheter
2 hr
Cardiac arrest
Grave ketosis at start of surgery
2 hr
Cardiac arrest
Grave ketosis at start of surgery
3 hr
Hemorrhage due to poor liver function
Cardiac arrest during surgery resulting in prolonged hypotension with bad perfusion of liver graft
6
5 Days
Intestinal obstruction
Excellent function of liver-pancreas graft until death
7
6 Days
Respiratory insufficiency due to bilateral hydrothorax
Excellent function of liver-pancreas graft until death
8
6 Days
Small bowel perforation and peritonitis
Excellent function of liver-pancreas graft until death
9
22 Days
Intestinal obstruction
Excellent function of liver-pancreas graft until death
110
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979
160 2 E E 120
I
:it : 2 ;L 2
----, 00
,/-I I
e-
,’ /’ /’
I
3
40
4
-1 : z
2
h, I 10
20
I 30
40
I 30
1 60
I 70
MlllUl~,
FIG. 2. Changes in mean arterial pressure of 12 animals ingroups 4 and 5 during recipient operation, (mean + SD). (1) removal of liver after clamping of IVC and establishing a portojugular shunt; (2) liver perfused with portal blood; (3) cross-clamping of aorta for arterial anastomosis; (4) clamps off aorta; (5) clamps off infrahepatic IVC.
rhages. Two animals died intraoperatively after the anastomoses of the suprahepatic IVC and the portal vein had been accomplished and portal blood was allowed to perfuse the liver. Both animals had a cardiac arrest. It was obvious that the animals were generally in poor condition following the time-consuming pancreatectomy that preceded the liver-pancreas grafting, and their blood pressure was often low after prolonged handling of the gut. This was an undesirable and sometimes fatal feature, since the blood pressure fell again in the unhepatic phase (see Fig. 2) and the liver was finally insufficiently perfused with blood containing accumulated toxic metabolites from the gut. In group 5 recipients with streptozotocininduced diabetes, four out of nine animals survived transplantation between 5 and 22 days and had excellent liver-pancreas function throughout their lifetime. At postmortem, the pancreatic grafts had a normal appearance without surrounding fluid collection, edema, or fibrosis. The p-cells had an almost normal histological appearance (see Figs. 3 and 4). Four pigs in this group died in the early postoperative period, which reflects their poor health due to the untreated diabetes induced between 5 and
11 days preoperatively. Once the potential diabetic recipients were subjected to a strict preoperative treatment, with a correction of the prevailing ketosis and fluid and electrolyte disorders, early postoperative deaths no longer occurred. While animals in group 4 recovered slowly from the side effects of total pancreatectomy and seldom started to feed during the first 2-3 days postoperatively, group 5 recipients were usually alert and in good condition on the day after surgery. Surviving pigs in groups 4 and 5 had a conspicuous consumption of glucose and potassium immediately after transplantation which had to be replaced accordingly. DISCUSSION This study shows that the phenomenon of survival of non-duct-ligated porcine pancreas allografts, in the presence of orthotopic liver allografts as described by Calne et al. [6, 221, can also be extended to ductligated pancreatic grafts. The mechanisms underlying the survival of pig liver allografts [4,24] and the donor-specific tolerogenic effect they exert in their new hosts are still obscure. The described technique of transplanting liver and duct-ligated pancreas en bloc in an orthotopic position is original and simple and resembles, in part, conventional liver transplantation in pigs 14, 231. In group 4 recipient animals, the blood pressure was already low after the timeconsuming pancreatectomy preceding transplantation and it fell further in the unhepatic phase leading to undesirable effects such as an insufficiently perfused liver graft and an accumulation in the blood of toxic metabolites from the gut. This led us to change from total pancreatectomy to the intravenous injection of streptozotocin for the induction of diabetes, a procedure which had several advantages. First, the recipient operation was much shorter and the animals were in better condition, presenting a higher blood pressure in the very critical period of initial perfusion with
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
111
FIG. 3. Islet from normal pig pancreas. Gomori, x375.
portal blood of the newly transplanted liver. Second, the duodenum was not skeletonized and surviving pigs could, therefore, feed the day after surgery, whereas recipient animals in group 4 with total pancreatectomy usually took 2-3 days to recover,
during which period they had to be provided with intravenous alimentation. Furthermore, the presence of the hosts’ own streptozotocin-damaged pancreas, which retained normal exocrine function, was advantageous in this model designed for a
FIG. 4. Islet from transplanted pancreas, pig No. 7 in group 5. Almost normal appearance of islet cells. Gomori, x375.
112
JOURNAL OF SURGICAL RESEARCH: VOL. 27, NO. 2, AUGUST 1979
long-term study of the grafts, since the animals needed no exocrine substitution. Howpigs were ever, streptozotocin-diabetic high-risk recipients and survived the lengthy transplant operation only after the adoption of a thorough preoperative assessment and regulation of their diabetic disorders. This factor was not realized during the first five experiments in group 5 and a series of inexplicable cardiac arrests ensued, which were absent in the latter part of the series. This study comprises too few experiments to be conclusive. However, rejection of pancreas grafts did not occur in the presence of liver allografts during the lifetime of the animals. We shall pursue this study in order to obtain long-term surviving pigs. Such animals could be useful for an analysis of endocrine pancreatic graft function in the absence of immunosuppression. ACKNOWLEDGMENT The authors thank Mrs. M. Oeltzschner, Anatomy Department, McGill University, Montreal, Canada, for the diagram.
REFERENCES 1. Ballinger, W. F., and Lacy, P. E. Transplantation of intact pancreatic islets in rats. Surgery 12: 175, 1972. 2. Bergan, J. J. ACSINIH Organ Transplant Reg. Newslett. 16, April 1976. 3. Bitter-Suermann, H., and Save-Soderbergh, J. The course of pancreas allografts in rats conditioned by spleen allografts. Transplantation 26: 28, 1978. 4. Calne, R. Y., White, H. J. O., Yoffa, D. E., Maginn, R. R., Binns, R. M., Samuel, J. R., and Molina, V. P. Observations of orthotopic liver transplantation in the pig. &if. Med. 1. 2: 478, 1%7. 5. Calne, R. Y., Sells, R. A., Pena, J. R., Davis, D. R., Millard, P. R., Herbertson, B. M., Binns, R. M., and Davies, D. A. L. Induction of immunological tolerance by porcine liver allografts. Nature (London) 223: 472, 1969. 6. Calne, R. Y., Sells, R. A., Marshall, V. C., Millard, P. R., Herbertson, B. M., Hadjiyannakis, E. J., Dunn, D. C., Robson, A. J., and Davis, D. R. Multiple organ grafts in the pig. Techniques and results of pancreatic, hepatic, cardiac and renal allografts. Brir. J. Surg. 59: 969, 1972.
7. de Cruyl, J., Westbroek, D. L., Dijkhuis, C. M., Vriesendorp, H. M., MacDicken, I., Elion-Gerritsen, W., Verschoor, L., Hulsmans, H. A. M., and Horchner, P. Influence of DL-A matching, ALS and 24-hour preservation on isolated pancreas allograft survival. Transplant. Proc. 5: 755, 1973. 8. Delmonico, F. L., Chase, C. M., and Russell, P. S. Transplantation of rat islets of Langerhans into diabetic mice. Transplant. Proc. 9: 367, 1977. 9. Frangipane, L. G., Poole, T. W., Barker, C. F., and Silvers, W. K. Vulnerability of allogeneic and xenogeneic pancreatic islets to alloantisera. Transplant. Proc. 9: 371, 1977.
lo. Gabel, H., Brynger, H., Bitter-Suermann, H., and
Henriksson, C. Surgically and chemically induced diabetes in pigs. Eur. Surg. Res. (Suppl. 1) 9: 285, 1977. 11. Hamilton, B., and Stein, A. F. The measurement of intravenous blood sugar curves. J. Lab. Clin. Med. 27: 491, 1942. 12 Kemp, C. B., Knight, M. J., Sharp, D. W., Bal’ linger, W. F., and Lacy, P. E. Effect oftransplantation site on results of pancreatic islet isografts in diabetic rats. Diabefologia 9: 486, 1973. 13. Kyriakides, G. K., Arora, V. K., Lifton, J., Nuttall, F. Q., and Miller, J. Porcine pancreatic transplantation. I. Autotransplantation of duct ligated segments. J. Surg. Res. 20: 451, 1976. 14. Kyriakides, G. K., Arora, V. K., Lifton, J., Nuttall, F. Q., and Miller, J. Porcine pancreatic transplants. II. Allotransplantation of duct ligated segments. J. Surg. Res. 20: 461, 1976. 15. Matas, A. J., Sutherland, D. E. R., and Najarian, J. S. Current status of islet and pancreas transplantation in diabetes. Diabetes 25: 785, 1976. 16. Najarian, J. S., Sutherland, D. E. R., Matas, A. J., Steffes, M. W., Simmons, R. L., and Goetz, F. C. Human islet transplantation: A preliminary report. Transplant.
Proc. 9: 233, 1977.
17. Nash, J. R., Peters, M., and Bell, P. R. F. Studies on the enhancement of rat islet allografts. Transplantation 25: 180, 1978. 18. Nelken, D., Friedman, E. A., Morse, S. I., and Beyer, M. M. Islet of Langerhans allotransplantation in the rat. Transplant. Proc. 9: 333, 1977. 19. Orloff, M. J., Lee, S., Charters, A. C., Grambort, D. E., Storck, L. G., and Knox, D. Long term studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg. 182: 198, 1975. 20. Reckard, C. R., and Barker, C. F. Transplantation of isolated pancreatic islets across strong and weak histocompatibility barriers. Transplant. Proc. 5: 761, 1973. 21. Reemtsma, K., Giraldo, N., Depp, D. A., and Eichwald, E. J. Islet cell transplantation. Ann. Surg. 168: 436, 1968. 22. Sells, R. A., Calne, R. Y., Hadjiyannakis, E. J.,
BITTER-SUERMANN
ET AL.: TRANSPLANTATION
OF LIVER AND PANCREAS
113
and Marshall, V. C. Glucose and insulin metabolism after pancreatic transplantation. Brir. Med. J.
survival of vascularized organ allografts in unimmunosuppressed pigs. Transplant. Proc. 5: 749,
3: 678, 1972. 23. Terblanche, J., Peacock, J. H., Bowes, J., and
1973. 25. Ziegler, M. M., Reckard, C. R., and Barker, C. F.
Hobbs, K. E. F. The technique of orthotopic liver homotransplantation in the pig. J. Surg. Res. 8: 151, 1968. 24. Terblanche, J., Hickman, R., Shippel, R. M., Dent, D. M., Spilg, H., Immelman, E. J., Du Toit, E., Uys, C. J., and Saunders, S. J. Prolonged
Long-term metabolic and immunological consideration in transplantation of pancreatic islets. J. Surg. Res. 16: 575, 1974. 26. Zimmermamt, G., Boeckl, O., Hell, E., and Seidl, P. Die iliakale Duodenopankreas Allotransplantation am Schwein. Chirurg 43: 372, 1972.
