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Long-term function of duct-ligated and free-duct whole pancreas transplants
JOURNAL
OF SURGICAL
RESEARCH
Long-Term KAZUNARI MARIANNE
35,283-292 (1983)
Function of Duct-Ligated and Free-Duct Whole Pancreas Transplants’
SATAKE, M.D., MARK A. HARDY, WOLFF, M.D., KEITH REEMTSMA,
M.D., MATTHEW J. NAGORSKY, M.D., M.D., AND ROMAN NOWYGROD, M.D.
Departments of Surgery and Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032
Submitted for publication November 26, 1982 This report compares endocrine and exocrine responses of “free-duct” and “duct-ligated” whole pancreas isografts in Lewis diabetic rats during a l-year period of observation. Fasting blood sugar, serum amylase, serum insulin, pancreozymin-secretin test, and L-arginine tolerance test assayswere performed prior to and at periodic intervals after transplantation. Histological examinations of transplanted and native pancreaswere performed monthly on animals in each group. Whole pancreastransplantations led to immediate and sustained normalization of FBS and insulin levels. In the “duct-ligated” group there was a gradual but insignificant decreaseof serum insulin levels after 9 months. Depressedserum amylase levels in diabetic rats were reversed in both groups. Isolated islet iso- and allografts also reversed depressedamylase levels. The results of pancreozymin-secretin tests in islet isogratkd animals supported the postulated trophic effect of endocrine pancreas on exocrine pancreas.Although both whole-pancreas transplant groups showed a normal response to GTT for 12 months after transplantation, the “ductligated” group showed a significantly lower response to L-arginine tolerance test than the “free duct” model as early as 4 months after transplantation, suggesting a defect in B-cell function in long-term “duct-ligated” graft. Serial histological examinations of transplanted pancreasesrevealed more rapid atrophy and more intense fibrosis of acinar tissue in the “duct-ligated” group than in the “free duct” group. These results suggestthat a “free duct” pancreatic isograft maintains a more stable endocrine function in a long-term study than does the “duct-ligated” pancreas, and that exocrine dysfunction in diabetic rats may be related to the endocrine disorder which can be reversed by both islet cell and whole-organ pancreatic transplantation.
grafts without duodenum are attractive because of the simplicity of the operative proIt has been well-established in experimental studiesthat isolated islets and whole/segmental cedure and the absenceof allogeneic intestinal tissue. In rats, Lee et al. have demonstrated pancreas transplantation can ameliorate or long-term normal endocrine function with this cure diabetes mellitus 19, 10, 14, 23, 27, 28, model despite the histologic appearance ofde30, 341. Although isolated islet cell implanterioration of islet cells and acinar fibrosis [20]. tation is attractive because of its simplicity, Recent reports in both experimental and clinsuccessful islet implantation has been hamical whole or segmental pancreatic transplanpered by problems with islet isolation and setation have described early graft failure due vere rejection problems [6, 26, 281. Recent to the leakageof amylase-rich pancreatic juice reports suggest that vascularized whole/segwith secondary severe pancreatitis, necrosis, mental pancreatic grafts are lessimmunogenic abscess,and sepsis[ 12-14, 16, 171.Studies in than isolated islets [25, 26, 28, 311 but mancanine, porcine, and clinical models suggest agement of the pancreatic duct and consethat intraperitoneal transplantation, leaving quent pancreatitis have led to major problems the duct free in the abdominal cavity, is a in clinical application of this method [4, 9, relatively safe and efficacious alternative to 11, 131. “Duct-ligated” or “blocked-duct” duct anastomosis or ligation [ 15, 17, 301.The present study was designed to compare long’ Supported by NIH Grants HL 14799-09 and AM term endocrine function in “free duct” and 196.52-05. INTRODUCTION
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“duct-ligated” models to determine which method is more effective in whole pancreas transplantation in the experimental rat model. Our previous report demonstrated the reversal of depressed serum amylase levels by whole pancreas transplantation in streptozotocin-induced diabetic rats and suggestedthat exocrine function is affected by endocrine function [29]. Exocrine dysfunction is present in 30% of all diabetic patients and in more than 60% of juvenile onset diabetic patients as indicated by the decreaseof output of bicarbonate, trypsin, and amylase when measured in vivo by duodenal aspiration following intravenous injection of secretin and CCKPZ [5, 7, 8, 331. A decreasein exocrine pancreatic function similar to that found in diabetic patients is seen in spontaneous or chemically induced diabetes in rats. The decrease, as measured by in vitro technique, is less than 20% of the level found in normal animals [ 1, 3, 241. Similar results were obtained with an in vivo assay using a guinea pig model [2]. In this study, therefore, we also attempted to determine the trophic effect of endocrine pancreas (the graft) on exocrine function of the host pancreas in vivo, using pancreozymin-secretin tests and serum amylase levels before and after grafting.
VOL. 35, NO. 4, OCTOBER
1983
vascular heterotopic pancreastransplantation method reported by Sun Lee et al, was developed to minimize graft handling and trauma [20]. In rats, pancreatic juice is excreted into bile ducts which open into the duodenum. The “free duct” grafts were prepared by leaving the common bile duct free in the abdominal cavity without any manipulation of the duct after the pancreatic head had been dissected free from the duodenum. End-to-side anastomoseswere performed using continuous 8-O nylon sutures between graft and host aorta and between graft portal vein and host inferior vena cava. The “ductligated” grafts were prepared by a technique similar to that of “free duct” grafts except that bile ducts were ligated with 6-O silk. The graft ischemic time was approximately 25 min. Technical success rate was 85% in both models. (B) “Hand-picked” islet cell implantation. In pancreatic islet iso- and allograft experiments we isolated islets by the technique of Lacy and Kostianowski using Percoll instead of Ficoll separation [ 181 and followed by “hand-picking.” Islets for iso- (800-900) and allografts ( 1OOO-1200) were injected intraportally into stable diabetic Lewis rats. Assays
MATERIALS
AND METHODS
Blood glucosedeterminations were done by the glucose oxidase technique immediately afAnimals ter collection of tail vein samples (in the early Inbred Lewis (RTl) and Wistar Furth (RTl) afternoon, following a 4-hr fast). rats weighing approximately 300 g were purPosttransplant determinations of serum chased from Microbiological Associates amylase were done using tail vein blood from (Walkersville, Md.). Diabetes was induced by at least six animals in each of the following intravenous administration of streptozotocin groups: Group 1, nontransplanted and anes(60 mg/kg). Only those animals that had fastthetized normal rats (n = 6); Group 2, ing blood glucose levels greater than 300 mg% nontransplanted, anesthetized normal rats for at least 2 weeks after injection of strep(n = 6); Group 3, diabetic rats (n = 6); Group tozotocin were usedin this study. Wistar Furth 4, diabetic recipients of “duct-ligated’ panrats served as donors for “hand-picked” islet creatic isografts (n = 10); Group 5, diabetic cell implantation. recipients of “free duct” pancreatic isografts (n = 10); Group 6, diabetic rats treated with Transplantation Technique daily insulin (2.5 u/day) injections for 7 days (A) Wholepancreas transplantation. A “no- prior to implantation of “hand-picked” islet touch” technique modification of the micro- allografts. Serum amylasedeterminations were
SATAKE
ET AL.: LONG-TERM
FUNCTION
performed at 2, 4, 6, 10, and 20 days and monthly thereafter following whole pancreas transplantation. In islet iso- and allografts, serum amylase was checked daily after grafting, for 2 weeks in isografted animals, and until blood sugar levels became elevated in allografted animals. Serum insulin determinations were performed by a double-antibody radioimmunoassay technique and were evaluated monthly following whole pancreas transplantation for more than 1 year.
OF PANCREAS
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after grafting. Sections of pancreas were stained both with hematoxylin and eosin with Gomori’s aldehyde fuchsin to evaluate the Bcells in the islets of Langerhans as well as the acinar components. The sections were interpreted by one of us (M.W.) without prior knowledge of the source of the pancreatic graft. Pancreozymin-Secretin Tests
Fasted animals were anesthetized with intraperitoneal chloral hydrate prior to formation of a blind duodenal loop for collection of pancreatic juice. This was made by ligation Glucose Tolerance Test of the proximal duodenum (just distal to the Animals were fasted for 3 hr and were anes- entry of a 17-gauge catheter into the duothetized with ether. They were given 1.5 g/kg denum). The catheter was inserted in the secof 50% glucose via the penile vein. Blood glu- ond portion of duodenum with its tip located cosedeterminations were done at 0, 5, 15, 30, at the papilla of Vater. Four injections of pan60, and 90 min after injection using the glucose creozymin-secretin (PZ 4.5u, Secretin 5.04~) oxidase technique. K values were evaluated at were given at 50-min intervals through a 231, 4, 8, and 12 months after transplantation gauge silastic catheter in the femoral vein. Pancreatic juice was collected for 30 min folusing the Lundback formula [22]. lowing the fast injection and the volume and amylase concentration were evaluated in the L-Arginine Tolerance Test following groups of animals: Group 1, nonL-Arginine insulin releasestimulation tests transplanted normal rats (n = 10); Group 2, were performed to determine the posttran- diabetic rats (n = 10); Group 3, diabetic splant function of B-cells in the following recipients of “hand-picked” islet isografts groups of six animals each at 1, 4, and 8 (n = 6). months after transplantation: Group 1, nontransplanted normal rats; Group 2, nontranRESULTS splanted diabetic rats; Group 3, diabetic recipients of “duct-ligated” pancreatic isografts; Whole Pancreas Transplantation Group 4, diabetic recipients of “free duct” (1) Fasting blood glucose and glucose tolpancreatic isografts. Animals fasted for 18 hr, erance test (Fig. 1). In all recipients of whole were anesthetized with ether and chloral hypancreas, blood glucose returned to normal drate, and were then injected with L-arginine levels by 10 hr after transplantation and re( 100 mg/kg) via the penile vein. The injections mained normal throughout the study period. were given over a 1-min time period. Femoral Control of hyperglycemia was equally effective vein samples were obtained 0, 3, 10, and 30 whether the duct was ligated or not. There min after injection. Serum insulin assayswere was no significant difference between the two performed by a double-antibody radioimgroups at any time during the study. All transmunoassay technique. planted animals showed a normal responseto the GTT at 8 months after transplantation. Morphologic Studies There were no significantly different K values Histologic examinations were performed on in the two models. biopsies of “duct-ligated” and “free duct” (2) Serum insulin (Fig. 2). In all diabetic grafts at 3 weeks and 1, 4, 8, and 12 months control rats, there was a significant fall in
286
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1983
-0- diabetic
(K--0.51) normal (K=2.27) free-duct (K 12.60 1 duct -ligated (K: 2.37)
-+ -A..-U-
600 1
50%
glucose
(l.Sg/kg)
I I I 1 30 60 90 Minutes FIG. I. Results of intravenous ghrcosetolerance test performed in different groups of diabetic, converted, and normal rats using 1.5 g/kg of 50% glucose 8 months after transplantation of “duct-ligated” or “free duct” pancreas and controls as indicated in the legend. L
I 15
1 5
1 0
normal (N ~10); 2.88-4.5 (plJ/L) free
< a
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30
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transplant
diabetic 4.25
(N : 10); 52.2 (JIU/L)
20
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Prior t0 Transplant
,
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,
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1
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4
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6
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Post
12
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FIG. 2. Results of insulin levels measured on a monthly basis in diabetic animals receiving “free duct” (0 - - - 0) or “duct-ligated” (0 ~ 0) pancreas grafts. The lower hatched line represents the almost absent insulin levels in the streptozotocin-induced diabetes and the upper hatched line representsthe insulin levels in normal animals folIowed concurrently for 1 year.
287
SATAKE ET AL.: LONG-TERM FUNCTION OF PANCREAS TRANSPLANTS
4 days). Eight to nine months following transplantation, serum amylase levels gradually declined in proportion to serum insulin levels in both models. “Free duct” transplant recipients had higher serum amylase levels than “duct-ligated” pancreas recipients after the initial postoperative period but the differences were not statistically significant. (4) L-Arginine insulin release stimulation test (Fig. 4). Normal rats showed a good responseto the L-arginine with a peak in insulin levels at 3 min which declined to normal by 30 min. No responseto the L-arginine was observed in diabetic rats. Both “free duct” and “ductligated” transplanted groups responded normally at 1 and 4 months after grafting. There was no significant response to the L-arginine observed at 8 months after transplantation in “duct-ligated” grafts while “free duct” pancreas recipients had a lower but still easily measurable response at 3 min after administration of L-arginine. (5) Histology (Fig. 5). In the “free duct” model, deterioration of the acinar component first appeared approximately 2 months after transplantation as evidenced by atrophy of the acinar tissue and duct ectasia. By 6 months
serum insulin levels (P < 0.001) as compared to normals. Both “duct-ligated” and “free duct” groups showed immediate normalization of insulin levels which persisted.The “free duct” transplant recipients had significantly higher insulin levels 9 months postoperatively than normal controls (P < 0.01) and insignificantly higher serum insulin levels than “duct-ligated” transplant recipients up to 1 year after transplantation. (3) Serum amylase (Fig. 3). There was no significant difference in serum amylase levels between nonanesthetized and anesthetized normal rats. Amylase levels of anesthetized normal rats served as control values. Diabetic rats had significantly lower amylase levels than controls (P < 0.001). “Free duct” transplant recipients initially showed significantly higher serum amylase levels than controls (P < 0.00 1 at 2 days, P < 0.05 at 4 days), but these returned to normal levels on Day 6 after transplantation and remained normal thereafter. By contrast, serum amylase levels did not show an initial rise in “duct-ligated” transplant recipients. The differences in the initial postoperative serum amylase levels between the two groups of pancreas recipients were highly significant (P < 0.001 at 2 days, P < 0.02 at
6000 I
normal
(N = 10);
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FIG. 3. Results of serial examination of amylase levels in whole pancreas isograft recipients showing no difference between “free duct” or “duct-ligated” isografts except in the first 5 days after transplantation.
288
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VOL. 35, NO. 4, OCTOBER 8 months
4 months
1 month I
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free duct (n =7) duct-ligated (n=7) normal (n:7) diabetic (n=7)
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FIG. 4. Results of L-arginine stimulation test at 1, 4, and 8 months in four groups of anesthetized rats as indicated in the legend. “Free duct” pancreas isograft recipients had more than the normal rise in insulin secretion at 3 min at 1 and 4 months and normal response at 8 months following transplantation, while “duct-ligated” pancreas recipients showed no response to L-arginine at 8 months.
FIG. 5. Photomicrograph of “free duct” pancreas isograft I 1 months after transplantation showing numerous concentration of islets Which are small and medium in size and have excellent granularity. Dilated acini and duct, as well as advanced fibrosis, are not shown well at this magnification (X80).
SATAKE
ET AL.: LONG-TERM
FUNCTION
after grafting, the acinar component became markedly atrophic and the tubules became smaller and more widely spaced. Fibrosis was not seen until 7 to 8 months after transplantation. Islets were well preserved, with good concentration, normal size, and excellent granularity until 8 months after transplantation. Thereafter, in most cases, there was a decreasein size and distortion of islets in proportion to the degree of fibrosis. “Duct-ligated” animals showed more rapid and intense fibrosis and atrophy of the acinar tissue than the “free duct” animals. Atrophy of the acinar tissue accompanied by duct ectasia began as early as 3 weeksafter transplantation. Fibrosis was first evident 4 to 5 months after transplantation
400.
OF PANCREAS
TRANSPLANTS
“Hand-picked” Islet Iso- and Allografts (1) Serum amylase and fasting blood glucose (Figs. 6 and 7). Daily injections of exogenousinsulin did not normalize blood sugar and amylase levels in the diabetic animals. Islet-isografted animals showed insulin and amylase elevation on Day 2 after grafting with a sustained return to normal levels 4 days after grafting. Fasting blood glucose and insulin levels remained normal in isograft recipients throughout the study. Islet allograft recipients (W/F to Lewis, rejection time 3-4 days) showed elevation of serum amylase levels in the initial period after grafting similar to that of islet isograft recipients. This was always followed by a decrease
Lewis
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289
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FIG. 6. Relationship between fasting blood sugar and serum amylase levels in diabetic animals that initially were treated with insulin and received syngeneic islets (900-1200) intraportally, leading to normalization of blood sugar and amylase levels.
290
JOURNAL OF SURGICAL RESEARCH: VOL. 35, NO. 4, OCTOBER 1983 40(
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FIG. 7. Relationship between blood sugar and amylase levels in diabetic recipients of W/F isolated islet allografis intraportally. Once islets were rejected on Day 4 after transplantation, serum amylase dropped precipitously.
in serum amylase levels once the animals rejetted the allograft and becamehyperglycemic.
showed normal pancreatic juice volume and amylase concentration.
(2) Pancreozymin-Secretintest (Table 1).
DISCUSSION Diabetic rats had significantly lower amylase concentrations and volumes of pancreatic Recently, Orloff et al. [27] have demonjuice than normal rats. Islet isografted animals strated that “duct-ligated” whole pancreas
TABLE I PANCREOZYMIN-SECRETIN TEST
(4 Normal rats Diabetic rats’ Pancreatic islet isograBsb
(8) (8) (5)
Volume (ml) (Mean + SD)
Amylase (IU/I) (Mean + SD)
1.16 + 0.27 0.69 f 0.22 1.24 f 0.30
29,485 + 17,237 104 + 82 21,687 f 7,521
0 Diabetes was induced with streptozotocin 55-65 mg/kg 4 weeks prior to study. ’ Isolated islet isografts consisted of 900-l 100 Lewis islets, given intraportally into diabetic rats.
SATAKEI
ET AL.: LONG-TERM
FUNCTION
transplantation in rats is as effective as pancreaticoduodenal transplantation [20] in controlling chemically induced diabetes. However, in both large-animal experiments and clinical segmental pancreas transplantation in the intraperitoneal position, numerous reports have demonstrated that severe postoperative complications frequently occur because of leakage of amylase-rich fluid from the peripancreatic area as a result of the intraductal back pressure from duct ligation [21]. We confirmed the observations of Kyriakides et al. [ 151and Toledo-Pereyra and Castellanos [32] that free intraperitoneal drainage of the pancreatic duct is well tolerated and safe becauseof the ability of the peritoneum to absorb the fluid. Important in our study was the observed difference between morphological changes and endocrine function following grafting with either “free duct” or “duct-ligated” pancreases.In the “free duct” group, islet cells were relatively well preserved throughout the study period. The ductal ectasia was first seen 2 months after transplantation when the acinar component became atrophic. These morphological findings suggestedthat exocrine function would gradually deteriorate. The “duct-ligated” group showed earlier duct ectasiathan that found in the “free duct” group and the acinar component became progressively fibrotic. Although islets were present throughout the study period in both groups, the degree of their deterioration was more marked in the “duct-ligated” than in the “free duct” grafts. Both the “duct-ligated” and “free duct” groups showed normal serum insulin levels and good responseto the GTT at all times during the study. Diabetic rats had significantly lower serum amylase levels than normal rats. Depressed amylase levels reversed in both models and remained normal throughout the study period regardless of histologic deterioration of the acinar component of the grafts and this may well be due to the persistent function of the native pancreas. This study also demonstrated in both models a different response to the L-arginine tolerance test at 8 months after transplantation
OF PANCREAS
TRANSPLANTS
291
despite normal GTT response. L-Arginine proved to be as good a stimulus as glucose for the releaseof insulin [27, 29, 321. Lambert et al. [ 191have suggestedthat stimulation of insulin release by amino acids depends upon the combined action of at least two components: (1) the metabolic activity of the /3 cell supplied either exogenously or endogenously and (2) the activity of receptor sites which may well prove to be the sites of insulin transport. Our results suggestthat the eventual decrease of insulin output by “duct-ligated” pancreatic isografts in response to L-arginine stimulation is related to the more severedegree of morphological changes of the islet cell quantity and probable membrane changes in “duct-ligated’ pancreatic grafts as compared to the “free duct” grafts. Moreover, serum amylase levels and pancreozymin-secretin testing in animals before and after islet cell implantation suggestthat exocrine pancreatic dysfunction in diabetic rats is at least in part secondary to the endocrine disorder and that the serum amylaselevel is not a good indicator for allograft rejection in the case of islet allografts. Recipients of either “free duct” or “duct-ligated” whole pancreases showed complete reversal to normal of the initially depressedserum amylase levels. The present study suggeststhat (1) “free duct” whole pancreas transplantation may be more effective and more applicable clinically than “duct-ligated” whole pancreas transplantation; (2) the L-arginine tolerance test is a very sensitive assay in determining the endocrine function of long-term pancreasgrafts; and (3) exocrine pancreatic dysfunction secondary to the endocrine disorder in clinical and experimental diabetes can be reversed by both isolated islet and whole pancreas transplantation. REFERENCES 1. Adler, G., and Kern, H. F. Regulation of exocrine pancreatic secretory process by insulin in vivo. Harm. Metab. Rex 7: 290, 1975. 2. Balk, M. W., Lang, C. M., and White, W. J. Exocrine pancreatic dysfunction in guinea pigs with diabetes mellitus. Lab. Invest. 32: 28, 1975. 3. Bazin, R., and Lavau, M. Diet composition and insulin
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effecton amylase to lipase ratio in pancreasof diabetic rats. Digestion 19: 386, 1979. 4. Bell, J. P., Salamonsen, L. A., Holland, G. W., et al. Autotransplantation of the pancreas in sheep;Insulin secretion from the transplant. J. Endocrinol. 48: 54, 1970. 5. Chey, W. Y., Shay, H., and Shuman, C. R. External pancreatic secretion in diabetes mellitus. Ann. Intern. Med. 59: 812, 1963. 6. Frangipane, L. G., Pode, T. W., Barker, C. F., and Silver, W. K. Vulnerability of allogeneic and xenogeneic pancreatic islets to antisera. Transplant. Proc. 9: 371, 1977. 7. Frier, B. M., Saunders,J. H., et al. Exocrine pancreatic function in juvenile onset diabetes mellitus. Gut 17: 685, 1976. 8. Floyd, J. C., Jr., Fajans, S. S., Pek, S., et al. Synergistic effect of certain amino acid pairs upon insulin secretion in man. Diabetes 19: 102, 1970. 9. Gliedman, M. L., Gold, M., Whittaker, J., et al. Pancreatic duct to ureter for exocrine drainage in pancreatic transplantation. Amer. J. Surg. 125: 245, 1973. 10. Grambort, D. E., Lee, S., et al. Pancreaticduct ligation and long-term endocrine function of pancreas transplants. Surg. Forum 24: 299, 1973. 11. Idezuki, Y., Feemster, J. A., Dietzman, R. H., and Lillehei, R. C. Experimental pancreatico-duodenal preservation and transplantation. Surg. Gynecol. Obstet. 126: 1002, 1968. 12. Kelly, W. O., Lillehei, R. C., Merkel, F. K., et al. Allotransplantation of pancreasand duodenum along with kidney in diabetic neuropathy. Surgery 61: 827, 1967. 13. Kyriakides, G. K., Arora, V. K., Lifton, J., et al. Porcine pancreatic transplantation I. Allotransplantation of “duct-ligated” segments. J. Surg. Rex 20: 451, 1976. 14. Kyriakides, G. K., Arora, V. K., Lifton, J., et al. Porcine pancreatic transplantation. II. Autotransplantation of “duct-ligated” segments.J. Surg. Res. 20: 461, 1976. 15. Kyriakides, G. K., Nutall, K., and Miller, J. Intraperitoneal segmental pancreatic ailografts with unligated duct in pigs. Transplant. Proc. 11: 527, 1979. 16. Kyriakides, G. K., Sutherland, D. E. R., Olson, L., et al. Segmental pancreatic transplantation in dogs. Transplant. Proc. 11: 530, 1979. 17. Kyriakides, G. K., Miller, J., Lifton, J., and Najarian, J. S. Effect of steroids on the structure and endocrine function of the “duct-ligated” porcine pancreatic autografts. Surg. Forum 15: 384, 1974. 18. Lacy, P. E., and Kostianovsky, M. D. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35, 1967.
19. Lambert, A. E., Kanazawa, Y., and Orci, L. Stimulation of insulin release in vitro by non-metabolized amino acid analogues. Proc. Sot Exp. Biol. Med. 137: 377, 1971. 20. Lee, S., Tung, K. S. K., Koopmass, H., Chandler, J. G., and Orloff, M. J. Pancreaticoduodenal transplantation in the rat. Transplantation 4: 421, 1972. 21. Lillehei, R. C., Simmons, R. L., Najarian, J. S., et al. Pancreaticoduodenal allotransplantation. Experimental and clinical experience. Ann. Surg. 172: 405, 1970. 22. Lundback, K. Intravenous glucose tolerance as a tool in definition and diagnosis of diabetes mehitus. Brit. Med. J. 2: 1507, 1962. 23. Merkel, F. K., ACS/NIH. Organ transplant registry. First scientific report. .I. Amer. Med. Assoc.217: 1520, 1971. 24. Mihier, R. D. G., and Hales, C. N. The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro. Diabetologia 3: 47, 1967. 25. Mori, S., Ota, K., Nabori, M., and Inou, T. Vascular changesin the pancreaticoduodenal allograft. Vusc. Surg. 2: 61, 1968. 26. Naji, A., Reckard, C. R., ZeigJer,M. M., and Barker, C. F. Vulnerability of pancreatic islets to immune cells and serum. Surg. Forum 26: 459, 1975. 27. Orloff, M. J., Lee, S., Charters, A. C., III, et al. Longterm studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg. 182: 198, 1975. 28. Perloff, L. J., Naji, A., Silvers, W. K., et al. Vascularized pancreas versus isolated islet allografts. An immunological comparison. Surgery 88: 222, 1980. 29. Satake, K., Nowygrod, R., Oluwole, S., et al. Free duct pancreatic transplantation in rats. Surg. Forum 30: 308, 1979. 30. Sutherland, D. E. R., Goez, F., and Najarian, J. S. Intraperitoneal transplantation of immediately vascularized segmental pancreatic grafts without duct ligation. Transplantation 28: 6, 1979. 31. Sutherland, D. E. R., Rynasiewicz, J. J., Kawahara, K., et al. Rejection of islets versus immediately vascularized pancreatic allografts. A quantitative comparison. J. Surg. Rex 29: 240, 1980. 32. Toledo-Pereyra, L. H., and Castellanos, J. Role of pancreatic duct ligation for segmental pancreas autotransplantation. Transplantation 2% 6, 1979. 33. Vacca, J. B., Henke, W. J., and Knight, W. A. The exocrine pancreas in diabetes mellitus. Ann. Intern. Med. 61: 242, 1964. 34. Weber, C., Zatriqui, A., McIntosh, R., et al. Pancreatic islet isografis, allografts and xenografts: Comparison of morphology and function. Surgery 79: 144, 1976.
OF SURGICAL
RESEARCH
Long-Term KAZUNARI MARIANNE
35,283-292 (1983)
Function of Duct-Ligated and Free-Duct Whole Pancreas Transplants’
SATAKE, M.D., MARK A. HARDY, WOLFF, M.D., KEITH REEMTSMA,
M.D., MATTHEW J. NAGORSKY, M.D., M.D., AND ROMAN NOWYGROD, M.D.
Departments of Surgery and Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032
Submitted for publication November 26, 1982 This report compares endocrine and exocrine responses of “free-duct” and “duct-ligated” whole pancreas isografts in Lewis diabetic rats during a l-year period of observation. Fasting blood sugar, serum amylase, serum insulin, pancreozymin-secretin test, and L-arginine tolerance test assayswere performed prior to and at periodic intervals after transplantation. Histological examinations of transplanted and native pancreaswere performed monthly on animals in each group. Whole pancreastransplantations led to immediate and sustained normalization of FBS and insulin levels. In the “duct-ligated” group there was a gradual but insignificant decreaseof serum insulin levels after 9 months. Depressedserum amylase levels in diabetic rats were reversed in both groups. Isolated islet iso- and allografts also reversed depressedamylase levels. The results of pancreozymin-secretin tests in islet isogratkd animals supported the postulated trophic effect of endocrine pancreas on exocrine pancreas.Although both whole-pancreas transplant groups showed a normal response to GTT for 12 months after transplantation, the “ductligated” group showed a significantly lower response to L-arginine tolerance test than the “free duct” model as early as 4 months after transplantation, suggesting a defect in B-cell function in long-term “duct-ligated” graft. Serial histological examinations of transplanted pancreasesrevealed more rapid atrophy and more intense fibrosis of acinar tissue in the “duct-ligated” group than in the “free duct” group. These results suggestthat a “free duct” pancreatic isograft maintains a more stable endocrine function in a long-term study than does the “duct-ligated” pancreas, and that exocrine dysfunction in diabetic rats may be related to the endocrine disorder which can be reversed by both islet cell and whole-organ pancreatic transplantation.
grafts without duodenum are attractive because of the simplicity of the operative proIt has been well-established in experimental studiesthat isolated islets and whole/segmental cedure and the absenceof allogeneic intestinal tissue. In rats, Lee et al. have demonstrated pancreas transplantation can ameliorate or long-term normal endocrine function with this cure diabetes mellitus 19, 10, 14, 23, 27, 28, model despite the histologic appearance ofde30, 341. Although isolated islet cell implanterioration of islet cells and acinar fibrosis [20]. tation is attractive because of its simplicity, Recent reports in both experimental and clinsuccessful islet implantation has been hamical whole or segmental pancreatic transplanpered by problems with islet isolation and setation have described early graft failure due vere rejection problems [6, 26, 281. Recent to the leakageof amylase-rich pancreatic juice reports suggest that vascularized whole/segwith secondary severe pancreatitis, necrosis, mental pancreatic grafts are lessimmunogenic abscess,and sepsis[ 12-14, 16, 171.Studies in than isolated islets [25, 26, 28, 311 but mancanine, porcine, and clinical models suggest agement of the pancreatic duct and consethat intraperitoneal transplantation, leaving quent pancreatitis have led to major problems the duct free in the abdominal cavity, is a in clinical application of this method [4, 9, relatively safe and efficacious alternative to 11, 131. “Duct-ligated” or “blocked-duct” duct anastomosis or ligation [ 15, 17, 301.The present study was designed to compare long’ Supported by NIH Grants HL 14799-09 and AM term endocrine function in “free duct” and 196.52-05. INTRODUCTION
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“duct-ligated” models to determine which method is more effective in whole pancreas transplantation in the experimental rat model. Our previous report demonstrated the reversal of depressed serum amylase levels by whole pancreas transplantation in streptozotocin-induced diabetic rats and suggestedthat exocrine function is affected by endocrine function [29]. Exocrine dysfunction is present in 30% of all diabetic patients and in more than 60% of juvenile onset diabetic patients as indicated by the decreaseof output of bicarbonate, trypsin, and amylase when measured in vivo by duodenal aspiration following intravenous injection of secretin and CCKPZ [5, 7, 8, 331. A decreasein exocrine pancreatic function similar to that found in diabetic patients is seen in spontaneous or chemically induced diabetes in rats. The decrease, as measured by in vitro technique, is less than 20% of the level found in normal animals [ 1, 3, 241. Similar results were obtained with an in vivo assay using a guinea pig model [2]. In this study, therefore, we also attempted to determine the trophic effect of endocrine pancreas (the graft) on exocrine function of the host pancreas in vivo, using pancreozymin-secretin tests and serum amylase levels before and after grafting.
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vascular heterotopic pancreastransplantation method reported by Sun Lee et al, was developed to minimize graft handling and trauma [20]. In rats, pancreatic juice is excreted into bile ducts which open into the duodenum. The “free duct” grafts were prepared by leaving the common bile duct free in the abdominal cavity without any manipulation of the duct after the pancreatic head had been dissected free from the duodenum. End-to-side anastomoseswere performed using continuous 8-O nylon sutures between graft and host aorta and between graft portal vein and host inferior vena cava. The “ductligated” grafts were prepared by a technique similar to that of “free duct” grafts except that bile ducts were ligated with 6-O silk. The graft ischemic time was approximately 25 min. Technical success rate was 85% in both models. (B) “Hand-picked” islet cell implantation. In pancreatic islet iso- and allograft experiments we isolated islets by the technique of Lacy and Kostianowski using Percoll instead of Ficoll separation [ 181 and followed by “hand-picking.” Islets for iso- (800-900) and allografts ( 1OOO-1200) were injected intraportally into stable diabetic Lewis rats. Assays
MATERIALS
AND METHODS
Blood glucosedeterminations were done by the glucose oxidase technique immediately afAnimals ter collection of tail vein samples (in the early Inbred Lewis (RTl) and Wistar Furth (RTl) afternoon, following a 4-hr fast). rats weighing approximately 300 g were purPosttransplant determinations of serum chased from Microbiological Associates amylase were done using tail vein blood from (Walkersville, Md.). Diabetes was induced by at least six animals in each of the following intravenous administration of streptozotocin groups: Group 1, nontransplanted and anes(60 mg/kg). Only those animals that had fastthetized normal rats (n = 6); Group 2, ing blood glucose levels greater than 300 mg% nontransplanted, anesthetized normal rats for at least 2 weeks after injection of strep(n = 6); Group 3, diabetic rats (n = 6); Group tozotocin were usedin this study. Wistar Furth 4, diabetic recipients of “duct-ligated’ panrats served as donors for “hand-picked” islet creatic isografts (n = 10); Group 5, diabetic cell implantation. recipients of “free duct” pancreatic isografts (n = 10); Group 6, diabetic rats treated with Transplantation Technique daily insulin (2.5 u/day) injections for 7 days (A) Wholepancreas transplantation. A “no- prior to implantation of “hand-picked” islet touch” technique modification of the micro- allografts. Serum amylasedeterminations were
SATAKE
ET AL.: LONG-TERM
FUNCTION
performed at 2, 4, 6, 10, and 20 days and monthly thereafter following whole pancreas transplantation. In islet iso- and allografts, serum amylase was checked daily after grafting, for 2 weeks in isografted animals, and until blood sugar levels became elevated in allografted animals. Serum insulin determinations were performed by a double-antibody radioimmunoassay technique and were evaluated monthly following whole pancreas transplantation for more than 1 year.
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after grafting. Sections of pancreas were stained both with hematoxylin and eosin with Gomori’s aldehyde fuchsin to evaluate the Bcells in the islets of Langerhans as well as the acinar components. The sections were interpreted by one of us (M.W.) without prior knowledge of the source of the pancreatic graft. Pancreozymin-Secretin Tests
Fasted animals were anesthetized with intraperitoneal chloral hydrate prior to formation of a blind duodenal loop for collection of pancreatic juice. This was made by ligation Glucose Tolerance Test of the proximal duodenum (just distal to the Animals were fasted for 3 hr and were anes- entry of a 17-gauge catheter into the duothetized with ether. They were given 1.5 g/kg denum). The catheter was inserted in the secof 50% glucose via the penile vein. Blood glu- ond portion of duodenum with its tip located cosedeterminations were done at 0, 5, 15, 30, at the papilla of Vater. Four injections of pan60, and 90 min after injection using the glucose creozymin-secretin (PZ 4.5u, Secretin 5.04~) oxidase technique. K values were evaluated at were given at 50-min intervals through a 231, 4, 8, and 12 months after transplantation gauge silastic catheter in the femoral vein. Pancreatic juice was collected for 30 min folusing the Lundback formula [22]. lowing the fast injection and the volume and amylase concentration were evaluated in the L-Arginine Tolerance Test following groups of animals: Group 1, nonL-Arginine insulin releasestimulation tests transplanted normal rats (n = 10); Group 2, were performed to determine the posttran- diabetic rats (n = 10); Group 3, diabetic splant function of B-cells in the following recipients of “hand-picked” islet isografts groups of six animals each at 1, 4, and 8 (n = 6). months after transplantation: Group 1, nontransplanted normal rats; Group 2, nontranRESULTS splanted diabetic rats; Group 3, diabetic recipients of “duct-ligated” pancreatic isografts; Whole Pancreas Transplantation Group 4, diabetic recipients of “free duct” (1) Fasting blood glucose and glucose tolpancreatic isografts. Animals fasted for 18 hr, erance test (Fig. 1). In all recipients of whole were anesthetized with ether and chloral hypancreas, blood glucose returned to normal drate, and were then injected with L-arginine levels by 10 hr after transplantation and re( 100 mg/kg) via the penile vein. The injections mained normal throughout the study period. were given over a 1-min time period. Femoral Control of hyperglycemia was equally effective vein samples were obtained 0, 3, 10, and 30 whether the duct was ligated or not. There min after injection. Serum insulin assayswere was no significant difference between the two performed by a double-antibody radioimgroups at any time during the study. All transmunoassay technique. planted animals showed a normal responseto the GTT at 8 months after transplantation. Morphologic Studies There were no significantly different K values Histologic examinations were performed on in the two models. biopsies of “duct-ligated” and “free duct” (2) Serum insulin (Fig. 2). In all diabetic grafts at 3 weeks and 1, 4, 8, and 12 months control rats, there was a significant fall in
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-0- diabetic
(K--0.51) normal (K=2.27) free-duct (K 12.60 1 duct -ligated (K: 2.37)
-+ -A..-U-
600 1
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glucose
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I I I 1 30 60 90 Minutes FIG. I. Results of intravenous ghrcosetolerance test performed in different groups of diabetic, converted, and normal rats using 1.5 g/kg of 50% glucose 8 months after transplantation of “duct-ligated” or “free duct” pancreas and controls as indicated in the legend. L
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1 0
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20
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4
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6
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FIG. 2. Results of insulin levels measured on a monthly basis in diabetic animals receiving “free duct” (0 - - - 0) or “duct-ligated” (0 ~ 0) pancreas grafts. The lower hatched line represents the almost absent insulin levels in the streptozotocin-induced diabetes and the upper hatched line representsthe insulin levels in normal animals folIowed concurrently for 1 year.
287
SATAKE ET AL.: LONG-TERM FUNCTION OF PANCREAS TRANSPLANTS
4 days). Eight to nine months following transplantation, serum amylase levels gradually declined in proportion to serum insulin levels in both models. “Free duct” transplant recipients had higher serum amylase levels than “duct-ligated” pancreas recipients after the initial postoperative period but the differences were not statistically significant. (4) L-Arginine insulin release stimulation test (Fig. 4). Normal rats showed a good responseto the L-arginine with a peak in insulin levels at 3 min which declined to normal by 30 min. No responseto the L-arginine was observed in diabetic rats. Both “free duct” and “ductligated” transplanted groups responded normally at 1 and 4 months after grafting. There was no significant response to the L-arginine observed at 8 months after transplantation in “duct-ligated” grafts while “free duct” pancreas recipients had a lower but still easily measurable response at 3 min after administration of L-arginine. (5) Histology (Fig. 5). In the “free duct” model, deterioration of the acinar component first appeared approximately 2 months after transplantation as evidenced by atrophy of the acinar tissue and duct ectasia. By 6 months
serum insulin levels (P < 0.001) as compared to normals. Both “duct-ligated” and “free duct” groups showed immediate normalization of insulin levels which persisted.The “free duct” transplant recipients had significantly higher insulin levels 9 months postoperatively than normal controls (P < 0.01) and insignificantly higher serum insulin levels than “duct-ligated” transplant recipients up to 1 year after transplantation. (3) Serum amylase (Fig. 3). There was no significant difference in serum amylase levels between nonanesthetized and anesthetized normal rats. Amylase levels of anesthetized normal rats served as control values. Diabetic rats had significantly lower amylase levels than controls (P < 0.001). “Free duct” transplant recipients initially showed significantly higher serum amylase levels than controls (P < 0.00 1 at 2 days, P < 0.05 at 4 days), but these returned to normal levels on Day 6 after transplantation and remained normal thereafter. By contrast, serum amylase levels did not show an initial rise in “duct-ligated” transplant recipients. The differences in the initial postoperative serum amylase levels between the two groups of pancreas recipients were highly significant (P < 0.001 at 2 days, P < 0.02 at
6000 I
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FIG. 3. Results of serial examination of amylase levels in whole pancreas isograft recipients showing no difference between “free duct” or “duct-ligated” isografts except in the first 5 days after transplantation.
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VOL. 35, NO. 4, OCTOBER 8 months
4 months
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FIG. 4. Results of L-arginine stimulation test at 1, 4, and 8 months in four groups of anesthetized rats as indicated in the legend. “Free duct” pancreas isograft recipients had more than the normal rise in insulin secretion at 3 min at 1 and 4 months and normal response at 8 months following transplantation, while “duct-ligated” pancreas recipients showed no response to L-arginine at 8 months.
FIG. 5. Photomicrograph of “free duct” pancreas isograft I 1 months after transplantation showing numerous concentration of islets Which are small and medium in size and have excellent granularity. Dilated acini and duct, as well as advanced fibrosis, are not shown well at this magnification (X80).
SATAKE
ET AL.: LONG-TERM
FUNCTION
after grafting, the acinar component became markedly atrophic and the tubules became smaller and more widely spaced. Fibrosis was not seen until 7 to 8 months after transplantation. Islets were well preserved, with good concentration, normal size, and excellent granularity until 8 months after transplantation. Thereafter, in most cases, there was a decreasein size and distortion of islets in proportion to the degree of fibrosis. “Duct-ligated” animals showed more rapid and intense fibrosis and atrophy of the acinar tissue than the “free duct” animals. Atrophy of the acinar tissue accompanied by duct ectasia began as early as 3 weeksafter transplantation. Fibrosis was first evident 4 to 5 months after transplantation
400.
OF PANCREAS
TRANSPLANTS
“Hand-picked” Islet Iso- and Allografts (1) Serum amylase and fasting blood glucose (Figs. 6 and 7). Daily injections of exogenousinsulin did not normalize blood sugar and amylase levels in the diabetic animals. Islet-isografted animals showed insulin and amylase elevation on Day 2 after grafting with a sustained return to normal levels 4 days after grafting. Fasting blood glucose and insulin levels remained normal in isograft recipients throughout the study. Islet allograft recipients (W/F to Lewis, rejection time 3-4 days) showed elevation of serum amylase levels in the initial period after grafting similar to that of islet isograft recipients. This was always followed by a decrease
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FIG. 6. Relationship between fasting blood sugar and serum amylase levels in diabetic animals that initially were treated with insulin and received syngeneic islets (900-1200) intraportally, leading to normalization of blood sugar and amylase levels.
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FIG. 7. Relationship between blood sugar and amylase levels in diabetic recipients of W/F isolated islet allografis intraportally. Once islets were rejected on Day 4 after transplantation, serum amylase dropped precipitously.
in serum amylase levels once the animals rejetted the allograft and becamehyperglycemic.
showed normal pancreatic juice volume and amylase concentration.
(2) Pancreozymin-Secretintest (Table 1).
DISCUSSION Diabetic rats had significantly lower amylase concentrations and volumes of pancreatic Recently, Orloff et al. [27] have demonjuice than normal rats. Islet isografted animals strated that “duct-ligated” whole pancreas
TABLE I PANCREOZYMIN-SECRETIN TEST
(4 Normal rats Diabetic rats’ Pancreatic islet isograBsb
(8) (8) (5)
Volume (ml) (Mean + SD)
Amylase (IU/I) (Mean + SD)
1.16 + 0.27 0.69 f 0.22 1.24 f 0.30
29,485 + 17,237 104 + 82 21,687 f 7,521
0 Diabetes was induced with streptozotocin 55-65 mg/kg 4 weeks prior to study. ’ Isolated islet isografts consisted of 900-l 100 Lewis islets, given intraportally into diabetic rats.
SATAKEI
ET AL.: LONG-TERM
FUNCTION
transplantation in rats is as effective as pancreaticoduodenal transplantation [20] in controlling chemically induced diabetes. However, in both large-animal experiments and clinical segmental pancreas transplantation in the intraperitoneal position, numerous reports have demonstrated that severe postoperative complications frequently occur because of leakage of amylase-rich fluid from the peripancreatic area as a result of the intraductal back pressure from duct ligation [21]. We confirmed the observations of Kyriakides et al. [ 151and Toledo-Pereyra and Castellanos [32] that free intraperitoneal drainage of the pancreatic duct is well tolerated and safe becauseof the ability of the peritoneum to absorb the fluid. Important in our study was the observed difference between morphological changes and endocrine function following grafting with either “free duct” or “duct-ligated” pancreases.In the “free duct” group, islet cells were relatively well preserved throughout the study period. The ductal ectasia was first seen 2 months after transplantation when the acinar component became atrophic. These morphological findings suggestedthat exocrine function would gradually deteriorate. The “duct-ligated” group showed earlier duct ectasiathan that found in the “free duct” group and the acinar component became progressively fibrotic. Although islets were present throughout the study period in both groups, the degree of their deterioration was more marked in the “duct-ligated” than in the “free duct” grafts. Both the “duct-ligated” and “free duct” groups showed normal serum insulin levels and good responseto the GTT at all times during the study. Diabetic rats had significantly lower serum amylase levels than normal rats. Depressed amylase levels reversed in both models and remained normal throughout the study period regardless of histologic deterioration of the acinar component of the grafts and this may well be due to the persistent function of the native pancreas. This study also demonstrated in both models a different response to the L-arginine tolerance test at 8 months after transplantation
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despite normal GTT response. L-Arginine proved to be as good a stimulus as glucose for the releaseof insulin [27, 29, 321. Lambert et al. [ 191have suggestedthat stimulation of insulin release by amino acids depends upon the combined action of at least two components: (1) the metabolic activity of the /3 cell supplied either exogenously or endogenously and (2) the activity of receptor sites which may well prove to be the sites of insulin transport. Our results suggestthat the eventual decrease of insulin output by “duct-ligated” pancreatic isografts in response to L-arginine stimulation is related to the more severedegree of morphological changes of the islet cell quantity and probable membrane changes in “duct-ligated’ pancreatic grafts as compared to the “free duct” grafts. Moreover, serum amylase levels and pancreozymin-secretin testing in animals before and after islet cell implantation suggestthat exocrine pancreatic dysfunction in diabetic rats is at least in part secondary to the endocrine disorder and that the serum amylaselevel is not a good indicator for allograft rejection in the case of islet allografts. Recipients of either “free duct” or “duct-ligated” whole pancreases showed complete reversal to normal of the initially depressedserum amylase levels. The present study suggeststhat (1) “free duct” whole pancreas transplantation may be more effective and more applicable clinically than “duct-ligated” whole pancreas transplantation; (2) the L-arginine tolerance test is a very sensitive assay in determining the endocrine function of long-term pancreasgrafts; and (3) exocrine pancreatic dysfunction secondary to the endocrine disorder in clinical and experimental diabetes can be reversed by both isolated islet and whole pancreas transplantation. REFERENCES 1. Adler, G., and Kern, H. F. Regulation of exocrine pancreatic secretory process by insulin in vivo. Harm. Metab. Rex 7: 290, 1975. 2. Balk, M. W., Lang, C. M., and White, W. J. Exocrine pancreatic dysfunction in guinea pigs with diabetes mellitus. Lab. Invest. 32: 28, 1975. 3. Bazin, R., and Lavau, M. Diet composition and insulin
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effecton amylase to lipase ratio in pancreasof diabetic rats. Digestion 19: 386, 1979. 4. Bell, J. P., Salamonsen, L. A., Holland, G. W., et al. Autotransplantation of the pancreas in sheep;Insulin secretion from the transplant. J. Endocrinol. 48: 54, 1970. 5. Chey, W. Y., Shay, H., and Shuman, C. R. External pancreatic secretion in diabetes mellitus. Ann. Intern. Med. 59: 812, 1963. 6. Frangipane, L. G., Pode, T. W., Barker, C. F., and Silver, W. K. Vulnerability of allogeneic and xenogeneic pancreatic islets to antisera. Transplant. Proc. 9: 371, 1977. 7. Frier, B. M., Saunders,J. H., et al. Exocrine pancreatic function in juvenile onset diabetes mellitus. Gut 17: 685, 1976. 8. Floyd, J. C., Jr., Fajans, S. S., Pek, S., et al. Synergistic effect of certain amino acid pairs upon insulin secretion in man. Diabetes 19: 102, 1970. 9. Gliedman, M. L., Gold, M., Whittaker, J., et al. Pancreatic duct to ureter for exocrine drainage in pancreatic transplantation. Amer. J. Surg. 125: 245, 1973. 10. Grambort, D. E., Lee, S., et al. Pancreaticduct ligation and long-term endocrine function of pancreas transplants. Surg. Forum 24: 299, 1973. 11. Idezuki, Y., Feemster, J. A., Dietzman, R. H., and Lillehei, R. C. Experimental pancreatico-duodenal preservation and transplantation. Surg. Gynecol. Obstet. 126: 1002, 1968. 12. Kelly, W. O., Lillehei, R. C., Merkel, F. K., et al. Allotransplantation of pancreasand duodenum along with kidney in diabetic neuropathy. Surgery 61: 827, 1967. 13. Kyriakides, G. K., Arora, V. K., Lifton, J., et al. Porcine pancreatic transplantation I. Allotransplantation of “duct-ligated” segments. J. Surg. Rex 20: 451, 1976. 14. Kyriakides, G. K., Arora, V. K., Lifton, J., et al. Porcine pancreatic transplantation. II. Autotransplantation of “duct-ligated” segments.J. Surg. Res. 20: 461, 1976. 15. Kyriakides, G. K., Nutall, K., and Miller, J. Intraperitoneal segmental pancreatic ailografts with unligated duct in pigs. Transplant. Proc. 11: 527, 1979. 16. Kyriakides, G. K., Sutherland, D. E. R., Olson, L., et al. Segmental pancreatic transplantation in dogs. Transplant. Proc. 11: 530, 1979. 17. Kyriakides, G. K., Miller, J., Lifton, J., and Najarian, J. S. Effect of steroids on the structure and endocrine function of the “duct-ligated” porcine pancreatic autografts. Surg. Forum 15: 384, 1974. 18. Lacy, P. E., and Kostianovsky, M. D. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35, 1967.
19. Lambert, A. E., Kanazawa, Y., and Orci, L. Stimulation of insulin release in vitro by non-metabolized amino acid analogues. Proc. Sot Exp. Biol. Med. 137: 377, 1971. 20. Lee, S., Tung, K. S. K., Koopmass, H., Chandler, J. G., and Orloff, M. J. Pancreaticoduodenal transplantation in the rat. Transplantation 4: 421, 1972. 21. Lillehei, R. C., Simmons, R. L., Najarian, J. S., et al. Pancreaticoduodenal allotransplantation. Experimental and clinical experience. Ann. Surg. 172: 405, 1970. 22. Lundback, K. Intravenous glucose tolerance as a tool in definition and diagnosis of diabetes mehitus. Brit. Med. J. 2: 1507, 1962. 23. Merkel, F. K., ACS/NIH. Organ transplant registry. First scientific report. .I. Amer. Med. Assoc.217: 1520, 1971. 24. Mihier, R. D. G., and Hales, C. N. The role of calcium and magnesium in insulin secretion from rabbit pancreas studied in vitro. Diabetologia 3: 47, 1967. 25. Mori, S., Ota, K., Nabori, M., and Inou, T. Vascular changesin the pancreaticoduodenal allograft. Vusc. Surg. 2: 61, 1968. 26. Naji, A., Reckard, C. R., ZeigJer,M. M., and Barker, C. F. Vulnerability of pancreatic islets to immune cells and serum. Surg. Forum 26: 459, 1975. 27. Orloff, M. J., Lee, S., Charters, A. C., III, et al. Longterm studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg. 182: 198, 1975. 28. Perloff, L. J., Naji, A., Silvers, W. K., et al. Vascularized pancreas versus isolated islet allografts. An immunological comparison. Surgery 88: 222, 1980. 29. Satake, K., Nowygrod, R., Oluwole, S., et al. Free duct pancreatic transplantation in rats. Surg. Forum 30: 308, 1979. 30. Sutherland, D. E. R., Goez, F., and Najarian, J. S. Intraperitoneal transplantation of immediately vascularized segmental pancreatic grafts without duct ligation. Transplantation 28: 6, 1979. 31. Sutherland, D. E. R., Rynasiewicz, J. J., Kawahara, K., et al. Rejection of islets versus immediately vascularized pancreatic allografts. A quantitative comparison. J. Surg. Rex 29: 240, 1980. 32. Toledo-Pereyra, L. H., and Castellanos, J. Role of pancreatic duct ligation for segmental pancreas autotransplantation. Transplantation 2% 6, 1979. 33. Vacca, J. B., Henke, W. J., and Knight, W. A. The exocrine pancreas in diabetes mellitus. Ann. Intern. Med. 61: 242, 1964. 34. Weber, C., Zatriqui, A., McIntosh, R., et al. Pancreatic islet isografis, allografts and xenografts: Comparison of morphology and function. Surgery 79: 144, 1976.