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The effect of warm ischemia and cold-storage preservation on rat pancreas transplantation
JOURNAL
OF SURGICAL
RESEARCH
36, 134- 139 (1984)
The Effect of Warm lschemia and Cold-Storage on Rat Pancreas Transplantation’ JAMES A. SCHULAK,
Preservation
M.D.,* AND JAMES KISTHARD
Department of Surgery, University of Iowa, College of Medicine, Iowa City, Iowa 52242
Submitted for publication May 10, 1983 Pancreatic isografts subjected to preharvest warm ischemia as well as cold-storage preservation in Collins’ solution were studied after transplantation into diabetic rats to determine whether warm &hernia will limit the ability to preserve pancreas grafts for transplantation. Warm ischemic periods of up to 2 hr did not alter islet function as measured by daily glucose levels and responseto intravenous glucose challenge. Likewise, hypothermic preservation of nonischemic pancreata was also well tolerated for up to 24 hr. However, the combination of preharvest warm &hernia and cold storage was deleterious. Whereas60 min of warm injury coupled with 12 hr of cold storageresulted in successfultransplantation in 86% of recipients, lengthening the duration of either warm or cold &hernia uniformly resulted in nonfunctioning grafts. Thus while islet function in the transplanted pancreas is very tolerant of warm &hernia alone, these studies suggestthat it should be kept to a minimum if cold storage preservation is to be used. MATERIALS
INTRODUCTION
AND
METHODS
Both Lewis and Lewis X Brown Norway Fl hybrid adult male rats were used in these experiments depending on their availability. In all casesdonors and recipients were syngeneic to each other thus ensuring that graft loss was due to either ischemia and preservation induced dysfunction or technical failure rather than immunologic rejection. Recipient rats were made hyperglycemic with streptozotocin (Upjohn) administered as an intravenous dose of between 55 and 65 mg/kg. Blood glucosewas measuredperiodically prior to transplantation and daily post-transplant with a reflectance calorimeter (Dextrometer, Ames) using a drop of tail capillary blood. This technique does not allow for accurate measurements of values greater than 400 mg/ dl; thus all such values are listed as >400. Only rats with stable diabetes whose blood glucose levels were consistently greater than 400 mg/dl were used as recipients. The technique of donor pancreatectomy ’ Supported in part by Biomedical Research Support and recipient transplantation has been deGrant RR 05372 from the Biomedical ResearchSupport Branch, Division of Research Facilities and Resources, scribed previously [l]. Briefly, the graft consisted of the segment of pancreas medial to National Institutes of Health. * To whom reprint requests should be addressed. the duodenum which was removed with the
It has been previously demonstrated that rat pancreatic islets can tolerate relatively long periods of pretransplant warm ischemia and retain their ability to control glucose homeostasiswhether transplanted either as primarily vascularized segmental grafts [l] or as intraportally injected isolated islets [2]. These observations suggest that the use of ischemic pancreata may be feasible in clinical transplantation providing that the ischemic insult does not either increase the likelihood of developing acinar pancreatitis or decrease the ability to successfully preserve the pancreas graft until transplantation can take place. The purpose of this study was to evaluate this latter problem by determining whether the combination of preharvest warm ischemia and pretransplant cold storagepreservation would adversely affect islet function in subsequently transplanted rat segmental pancreas isografts.
0022-4804/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All rights of reprcductmn in any form resewed.
134
SCHULAK AND KISTHARD:
135
PRESERVATION OF ISCHEMIC PANCREAS
celiac axis and portal vein. All grafts were ligated just proximal to the line of dissection thereby creating a duct-obstructed specimen. Recipient animals underwent midline laparotomy under chloral hydrate and ether anesthesia. Vascular continuity was reestablished in the recipient by anastomosis of the graft celiac axis and portal vein to the infrarenal aorta and vena cava using standard microsurgical techniques. These anastomoseswere performed in between 15 and 30 min in all animals. A warm ischemic insult was delivered to the donor pancreata prior to their removal. This was accomplished by partially dissecting out the graft and then sacrificing the donor by exsanguination. Final dissection of the graft was delayed until the designated amount of ischemic time was achieved. Since in our previous experiments [I], where we studied the effect of 37°C ischemia on the function of rat pancreas transplants, 90 min of &hernia was well tolerated, we began this study also with a 90-min period of ischemia. Just prior to excision, the organs were flushed through the aorta with a cold heparin-saline solution. Following harvest the pancreata were either immediately transplanted into a previously prepared recipient or placed into cold storage. Cold-storagepreservation wasaccomplished by placing the pancreata into a 50-ml plastic specimen container filled with standard intracellular electrolyte solution in the form of ice slush. In all experiments Collins’ solution was used to which 10 ml of 1% Xylocaine, 15 ml of MgS04, and 50 ml of 50% dextrose were added per liter. The containers were then capped and stored in a refrigerator at 5°C for the designated amount of preservation time. Both composition of the electrolyte solution and technique of preservation utilized in these experiments were similar to those used in our clinical kidney transplantation practice. Between 2 and 3 weeks after successful transplantation, all recipients underwent an intravenous glucose tolerance test (IVGTT) using 500 mg/kg of dextrose/animal. Glucose decay constants (k values) were calculated from the plotted curves using a standard for-
mula [3]. Graft pancreatectomy was performed several days after the IVGTTs. Subsequently, blood glucose values were again monitored daily until recurrent hyperglycemia was documented. Representative specimens were fixed in formalin, sectioned, and stained with hematoxylin and eosin for histological evaluation. RESULTS
The results of these experiments are summarized in Table 1. Both 90- and 120-min periods of in situ warm &hernia were well tolerated by the pancreatic grafts as manifested by a rapid amelioration of the diabetic state in all recipients (Table 2). Mean blood glucose values of 100 f 10 and 136 f 23 mg/dl (*SD) were observed at 2 weeks following transplantation for these two groups respectively (groups 1 and 2, Table 2). These values as well as those recorded prior to 2 weeks were not significantly different from those for the control, nonischemic rats using t test analysis. TABLE 1 GRAFT SURVIVAL DATA ACCORDING TO EXPERIMENTAL GROUP
Experimental group 1. Control-no &hernia 2. 90&n warm ischemia no cold storage 3. 120-min warm &hernia no cold storage 4. No warm &hernia 12-hr cold storage 5. No warm &hernia 24-hr cold storage 6. No warm &hernia 36-hr cold storage 7. No warm &hernia 48-hr cold storage 8. 60-min watm &hernia 12-hr cold storage 9. 60-min warm &hernia 24-hr cold storage 10. 120-min warm ischemia 6-hr cold storage II. 30-min warm ischemia 18-hr cold storage
NOllllOTotal glycemic” tramat 2 weeks planted
Percent S”ccess
3
3
100
3
3
100
4
4
100
4
5
80
4
5
80
2
4
50
0
2
0
6
7
86
0
4
0
0
4
0
0
2
0
’ Blood glucose of less than 150 mg/dl.
136
JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 2, FEBRUARY 1984 TABLE 2
BLEND GLUCOSEVALUES OVER THE COURSEOFTHE STUDY FORTHE SUCCESSFUL EXPERIMENTALGROW
Days after transplant Experimental groupa
1
2
4
8
15
Pan-X b
Mean blood glucose mg/dl (*SD) 1. Control no ischemia 2. 90-min warm ischemia no cold storage 3. 120-min warm ischemia no cold storage 4. No warm ischemia 12-hr cold storage 5. No warm ischemia 24-hr cold storage 6. No warm ischemia 36-hr cold storage 8. 60-min warm ischemia 12-hr cold storage
88 + 24’ 116 + 26
103 f 10
109 f
23
90 f 25
108 f 27
>400
ill?
108 f
27
112 f 33
92+ 12
>400
3
97f
7
143 + 40
128 + 28
121 f 19
136 + 23
>400
98?
19
130 f 32
140 f
56
115 + 32
120 * 40
>400
92?
12
138 + 31
112-c 27
131 f 49
116 2 21
>400
256 f 181
115 f 48
256 f 181
154 + 30
121 -c 6
1400
128 f
133 + 31
1OOf 11
118 -c 15
103 + 12
>400
35
’ Group numbers correspond to those in Table 1. b Postgraft pancreatectomy. ’ No statistically significant differences between any of the experimental values and their corresponding control value (t test).
IVGTT curves (Fig. 1) and k values of 3.0 and 3.5, respectively, were nearly identical for both of these groups revealing a normal, rapid decay of glucose following intravenous glucose challenge. Four groups of rats were studied after receiving pancreata preserved by cold storage in
400
090
-
MIN WARM
0120
ii
MIN WARM
ISCHEMIA ISCHEMIA
2
i_: 300
h
0
~
5
10
15
20
1 60
30 TIME
(mid
FIG. 1. IVGTT (dextrose 500 mg/kg) curves for recipients of pancreatic grafts subjected to preharvest warm &hernia.
Collins’ solution. Eighty percent of the recipients receiving grafts stored for either 12 or 24 hr developed normoglycemia (groups 4 and 5, Table 1). Glucose values for the course of the experiment for these two successful groups are also listed in Table 2 and are not significantly different from those obtained in the warm ischemic or control animals either. The one failed animal in the 24-h preservation group had a blood glucose of 16 mg/dl on the first postoperative day and was found dead 1 day later which suggests that islet dysfunction with massive insulin release may have occurred. Likewise, the one failure in the 12-hr preservation group was found dead on the first postoperative day, possibly also having suffered from fatal hypoglycemia as an autopsy did not reveal any other obvious cause of death. Ability to handle an intravenous glucose challenge was not impaired by the hypothermic preservation for either 12 or 24 hr as indicated by normal IVGTT curves (Fig. 2) and k constants of 2.8 and 3.8 for the surviving animals in these two groups.
SCHULAK
AND
KISTHARD:
012
HOUR
COLD
ISCHEMIA
0 24 HOUR
COLD
ISCHEMIA
400
18
0
2
0
1.
5
10
15
PRESERVATION
1
20
60
30 TIME
hid
FIG. 2. IVGTT (dextrose 500 mg/kg) curves for recipients of pancreatic grafts subjected to pretransplant hypothermic preservation in Collins’ solution.
In contrast, extension of the period of coldstorage preservation past 24 hr was not well tolerated by the pancreatic islets in these experiments. Only two of the four recipients of pancreata stored for 36 hr became normoglycemic at 2 weeks, while neither of the two recipients of a 48-hr stored graft regained normal glucose homeostasis (groups 6 and 7, Table 1). The addition of preharvest warm ischemia to the insult of cold-hypothermic preservation reduced the amount of cold-storage time tolerated by these rat pancreatic grafts. One hour of warm ischemia prior to preservation was well tolerated by pancreata cold stored for only 12 hr. In this group (group 7, Table 1) six of seven recipients became normoglycemic following transplantation and had a mean blood glucose of 103 f 12 mg/dl at 2 weeks. Again, this value was not significantly different from either controls or the other successful experimental groups. In addition, response to glucose challenge for this group (group 7) was identical to that observed in the 12-hr preservation-alone group (group 3) as manifested both by very similar IVGTT curves (Figure 3) and k values of 3.0 and 2.8, respectively. Also, daily glucose values following transplantation were not remarkably different from those of the other successfulgroups (Table 2). On the other hand, 60 min of preharvest warm ischemia coupled with 24 hr of hypothermic
OF ISCHEMIC
PANCREAS
137
preservation resulted in nonfunction of all four grafts so attempted (group 8, Table 1). Neither increasing the warm ischemia time to 120 min and decreasingcold-storagetme to 6 hr (group 9, Table 1) nor decreasing warm time to 30 min and increasing preservation time to 18 hr (group 10, Table 1) was successful. All of these grafts either failed to reverse the pretransplant hyperglycemia or resulted in recipient death, again presumably due to hypoglycemia. Graft pancreatectomy was followed by a prompt return of hyperglycemia in all recipients of grafts with functioning islets suggesting that the level of glucose homeostasis observed was indeed due to the presence of the pancreatic graft. Histological review of these specimens from the normoglycemic recipients revealed abundant islets with normal morphology (Figure 4). On the other hand, islets were sparse, if present at all, in the grafts of the rats that were unsuccessfully transplanted suggesting that excessive ischemia whether cold or warm can result in islet degeneration. Acinar degeneration, fat necrosis, ductal dilatation, and varying degreesof inflammatory infiltration were observed in the exocrine pancreas of all specimens and thus cannot be solely attributed to ischemic injury as discussed previously [ 11.
500 .12
IIOUR
COLD
ISCHEMIA
4 0
5
10
15
20
30
60
TIME (mid
FIG. 3. IVGTI
(dextrose 500 mg/kg) curve for recipients of pancreatic grafts subjected to both preharvest warm ischemia and pretransplant hypothermic preservation in Collins’ solution compared to the IVGTT curve for recipients of nonischemic, cold-stored grafts.
138
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 36, NO. 2, FEBRUARY
1984
FIG. 4. Photomicrograph of a pancreatic graft subjected to I hr of preharvest warm &hernia and 12 hr of hypothermic preservation in Collins’ solution taken from a group 8 recipient with normal post-transplant glucose homeostasis. Note abundant, normal appearing islets (arrows) and the marked degeneration of surrounding acinar tissue (H&E, X80).
DISCUSSION
Successful hypothermic preservation of canine pancreatic grafts in iced intracellular electrolyte solution has been reported previously by others [4, 51. Whether simple cold storage is a more efficacious technique for pancreas preservation than pulsatile machine preservation is still controversial [6]. However, data recently reported by Florack et al. [7] comparing cold storage of canine pancreata in Collins’ solution, modified silica-gel-filtered plasma, and machine perfusion were clearly in favor of the nonperfusion technique. In none of these studies, however, was the question of whether a prior warm ischemic insult will adversely affect the ability to preserve the grafts by cold storage addressed. While Westbroek et al. [8] reported that 30 min of warm &hernia did not appreciably alter the outcome of machine perfused canine grafts they did
not study its effect on those that were cold stored. In clinical kidney transplantation, it is generally believed that warm ischemia shortens the safe period for subsequent graft preservation and thus it might be expected to have a similar influence on pancreas preservation and transplantation. In this study we achieved two goals. First, we demonstrated successful preservation and transplantation of rat pancreatic grafts using iced Collins’ solution as the storage medium. Normal glucose homeostasis was achieved immediately following transplantation in most recipients of organs stored for up to 24 hr. Longer periods of hypothermia were apparently harmful to the pancreata as only 50% of those hypothermically stored for 36 hr functioned and then somewhat sluggishly early on as indicated by their greater blood glucose values immediately following transplantation. Only two attempts at 48-hr storage were made
SCHULAK AND KISTHARD:
PRESERVATION OF ISCHEMIC PANCREAS
but both of these were clearly unsuccessful. Our observation of endocrine dysfunction with greater than 24 hr of cold storage is similar to that reported for canine pancreata also stored in Collins’ solution [5]. Nevertheless, this experiment demonstrates the feasibility of achieving successful nonperfusion hypothermic preservation of rat pancreata and thereby presents a model for subsequent study of this problem. The second and more important aim of this study was to evaluate the effect of preharvest warm ischemia on subsequent cold-storage preservation of the pancreas. Our data suggest that such an ischemic insult experienced prior to cooling may indeed significantly reduce the preservation potential for long-term hypothermic storage of this organ. While either 120 min or less of warm ischemia or up to 24 hr of cold storage were routinely well tolerated by the pancreatic grafts a combination of the two usually resulted in islet dysfunction. Only 12 hr of preservation were possible when warm ischemia was limited to 60 min or less. Lengthening the preservation time and decreasing the amount of warm ischemia did not permit adequate islet function. Similarly, decreasing storage time and increasing warm ischemia also resulted in failed grafts. These preliminary studies do not suggesta mechanism for the observations described. It may be speculated, however, that an ischemic insult of the hypothermic variety is additive to the warm induced injury of pancreatic islets that we described previously [ 11.Thus certain combinations of the two result in a cumulative injury that exceedsthe ability of the islets to recover and results in their rapid degeneration. In support of this hypothesis of additive injury is our recent observation that up to 5 hr of ambient temperature ischemia alone, as used in this study, was well tolerated by transplant
139
pancreata in rats [9]. Lastly, whether or not hypothermic preservation of ischemic pancreatic grafts in media other than Collins’ solution may yield better results as has been suggestedfor kidneys [lo] is uncertain. Nevertheless, these experiments suggest that in clinical pancreas transplantation, an effort should be made to limit warm ischemic time to a minimum if the use of hypothermic preservation in Collins’ solution is contemplated. REFERENCES 1. Schulak, J. A., Franklin, W. A., Stuart, F. P., and Reckard, C. R. Effect of warm &hernia on segmental pancreas transplantation in the rat. Transplantation 35: 7, 1983. 2. Matas, A. J., Sutherland, D. E. R., Payne, W. D., Kretschmer, G. J., Steffes, M. W., and Najarian, J. S. The critical period of donor ischemia in neonatal rats. Transplantation 23: 295, 1977. 3. Williams, R. H., and Porte, D. The pancreas. In R. H. Williams (Ed.), Textbook of Endocrinology, Philadelphia: Saunders, 1974. P. 565. 4. Baumgartner, D., Sutherland, D. E. R., Heil, J. E., Zweber, B., Awad, E. A., and Najarian, J. S. Cold storage of segmental canine pancreatic grafts for 24 hours. J. Surg. Res. 29: 248, 1980. 5. Florack, G., Sutherland, D. E. R., Heil, J., Zweber, B., and Najarian, J. S. Long-term preservation of segmental pancreas autografts. Surgery 92: 260, 1982. 6. Toledo-Pereyra, L. H. Organ preservation. 1. Kidney and pancreas. J. Surg. Res. 30: 165, 1981. 7. Florack, G., Sutherland, D. E. R., Heil, J., Squifflet, J. P., and Najarian, J. S. Preservation of canine segmental pancreatic autografts: cold storage versus pulsame machine preservation. J. Surg. Res. 34: 493, 1983. 8. Westbroek, D. L., deGruy1, J., Digkhuis, C. M., et a/. Twenty-four-hour hypothermic preservation perfusion and storageof the duct-ligated canine pancreas with transplantation. Transplant Proc. 6: 319, 1974. 9. Schulak, J. A. Effect of post mortem ischemia on the function of adult rat islets following pancreatic transplantation. Metabolism 32: 643, 1983. 10. Halasz, N. A., and Collins, G. M. Forty-eight-hour kidney preservation. A comparison of flushing and ice storagewith perfusion. Arch. Surg. 111: 175, 1976.
OF SURGICAL
RESEARCH
36, 134- 139 (1984)
The Effect of Warm lschemia and Cold-Storage on Rat Pancreas Transplantation’ JAMES A. SCHULAK,
Preservation
M.D.,* AND JAMES KISTHARD
Department of Surgery, University of Iowa, College of Medicine, Iowa City, Iowa 52242
Submitted for publication May 10, 1983 Pancreatic isografts subjected to preharvest warm ischemia as well as cold-storage preservation in Collins’ solution were studied after transplantation into diabetic rats to determine whether warm &hernia will limit the ability to preserve pancreas grafts for transplantation. Warm ischemic periods of up to 2 hr did not alter islet function as measured by daily glucose levels and responseto intravenous glucose challenge. Likewise, hypothermic preservation of nonischemic pancreata was also well tolerated for up to 24 hr. However, the combination of preharvest warm &hernia and cold storage was deleterious. Whereas60 min of warm injury coupled with 12 hr of cold storageresulted in successfultransplantation in 86% of recipients, lengthening the duration of either warm or cold &hernia uniformly resulted in nonfunctioning grafts. Thus while islet function in the transplanted pancreas is very tolerant of warm &hernia alone, these studies suggestthat it should be kept to a minimum if cold storage preservation is to be used. MATERIALS
INTRODUCTION
AND
METHODS
Both Lewis and Lewis X Brown Norway Fl hybrid adult male rats were used in these experiments depending on their availability. In all casesdonors and recipients were syngeneic to each other thus ensuring that graft loss was due to either ischemia and preservation induced dysfunction or technical failure rather than immunologic rejection. Recipient rats were made hyperglycemic with streptozotocin (Upjohn) administered as an intravenous dose of between 55 and 65 mg/kg. Blood glucosewas measuredperiodically prior to transplantation and daily post-transplant with a reflectance calorimeter (Dextrometer, Ames) using a drop of tail capillary blood. This technique does not allow for accurate measurements of values greater than 400 mg/ dl; thus all such values are listed as >400. Only rats with stable diabetes whose blood glucose levels were consistently greater than 400 mg/dl were used as recipients. The technique of donor pancreatectomy ’ Supported in part by Biomedical Research Support and recipient transplantation has been deGrant RR 05372 from the Biomedical ResearchSupport Branch, Division of Research Facilities and Resources, scribed previously [l]. Briefly, the graft consisted of the segment of pancreas medial to National Institutes of Health. * To whom reprint requests should be addressed. the duodenum which was removed with the
It has been previously demonstrated that rat pancreatic islets can tolerate relatively long periods of pretransplant warm ischemia and retain their ability to control glucose homeostasiswhether transplanted either as primarily vascularized segmental grafts [l] or as intraportally injected isolated islets [2]. These observations suggest that the use of ischemic pancreata may be feasible in clinical transplantation providing that the ischemic insult does not either increase the likelihood of developing acinar pancreatitis or decrease the ability to successfully preserve the pancreas graft until transplantation can take place. The purpose of this study was to evaluate this latter problem by determining whether the combination of preharvest warm ischemia and pretransplant cold storagepreservation would adversely affect islet function in subsequently transplanted rat segmental pancreas isografts.
0022-4804/84 $1.50 Copyright 0 1984 by Academic Press, Inc. All rights of reprcductmn in any form resewed.
134
SCHULAK AND KISTHARD:
135
PRESERVATION OF ISCHEMIC PANCREAS
celiac axis and portal vein. All grafts were ligated just proximal to the line of dissection thereby creating a duct-obstructed specimen. Recipient animals underwent midline laparotomy under chloral hydrate and ether anesthesia. Vascular continuity was reestablished in the recipient by anastomosis of the graft celiac axis and portal vein to the infrarenal aorta and vena cava using standard microsurgical techniques. These anastomoseswere performed in between 15 and 30 min in all animals. A warm ischemic insult was delivered to the donor pancreata prior to their removal. This was accomplished by partially dissecting out the graft and then sacrificing the donor by exsanguination. Final dissection of the graft was delayed until the designated amount of ischemic time was achieved. Since in our previous experiments [I], where we studied the effect of 37°C ischemia on the function of rat pancreas transplants, 90 min of &hernia was well tolerated, we began this study also with a 90-min period of ischemia. Just prior to excision, the organs were flushed through the aorta with a cold heparin-saline solution. Following harvest the pancreata were either immediately transplanted into a previously prepared recipient or placed into cold storage. Cold-storagepreservation wasaccomplished by placing the pancreata into a 50-ml plastic specimen container filled with standard intracellular electrolyte solution in the form of ice slush. In all experiments Collins’ solution was used to which 10 ml of 1% Xylocaine, 15 ml of MgS04, and 50 ml of 50% dextrose were added per liter. The containers were then capped and stored in a refrigerator at 5°C for the designated amount of preservation time. Both composition of the electrolyte solution and technique of preservation utilized in these experiments were similar to those used in our clinical kidney transplantation practice. Between 2 and 3 weeks after successful transplantation, all recipients underwent an intravenous glucose tolerance test (IVGTT) using 500 mg/kg of dextrose/animal. Glucose decay constants (k values) were calculated from the plotted curves using a standard for-
mula [3]. Graft pancreatectomy was performed several days after the IVGTTs. Subsequently, blood glucose values were again monitored daily until recurrent hyperglycemia was documented. Representative specimens were fixed in formalin, sectioned, and stained with hematoxylin and eosin for histological evaluation. RESULTS
The results of these experiments are summarized in Table 1. Both 90- and 120-min periods of in situ warm &hernia were well tolerated by the pancreatic grafts as manifested by a rapid amelioration of the diabetic state in all recipients (Table 2). Mean blood glucose values of 100 f 10 and 136 f 23 mg/dl (*SD) were observed at 2 weeks following transplantation for these two groups respectively (groups 1 and 2, Table 2). These values as well as those recorded prior to 2 weeks were not significantly different from those for the control, nonischemic rats using t test analysis. TABLE 1 GRAFT SURVIVAL DATA ACCORDING TO EXPERIMENTAL GROUP
Experimental group 1. Control-no &hernia 2. 90&n warm ischemia no cold storage 3. 120-min warm &hernia no cold storage 4. No warm &hernia 12-hr cold storage 5. No warm &hernia 24-hr cold storage 6. No warm &hernia 36-hr cold storage 7. No warm &hernia 48-hr cold storage 8. 60-min watm &hernia 12-hr cold storage 9. 60-min warm &hernia 24-hr cold storage 10. 120-min warm ischemia 6-hr cold storage II. 30-min warm ischemia 18-hr cold storage
NOllllOTotal glycemic” tramat 2 weeks planted
Percent S”ccess
3
3
100
3
3
100
4
4
100
4
5
80
4
5
80
2
4
50
0
2
0
6
7
86
0
4
0
0
4
0
0
2
0
’ Blood glucose of less than 150 mg/dl.
136
JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 2, FEBRUARY 1984 TABLE 2
BLEND GLUCOSEVALUES OVER THE COURSEOFTHE STUDY FORTHE SUCCESSFUL EXPERIMENTALGROW
Days after transplant Experimental groupa
1
2
4
8
15
Pan-X b
Mean blood glucose mg/dl (*SD) 1. Control no ischemia 2. 90-min warm ischemia no cold storage 3. 120-min warm ischemia no cold storage 4. No warm ischemia 12-hr cold storage 5. No warm ischemia 24-hr cold storage 6. No warm ischemia 36-hr cold storage 8. 60-min warm ischemia 12-hr cold storage
88 + 24’ 116 + 26
103 f 10
109 f
23
90 f 25
108 f 27
>400
ill?
108 f
27
112 f 33
92+ 12
>400
3
97f
7
143 + 40
128 + 28
121 f 19
136 + 23
>400
98?
19
130 f 32
140 f
56
115 + 32
120 * 40
>400
92?
12
138 + 31
112-c 27
131 f 49
116 2 21
>400
256 f 181
115 f 48
256 f 181
154 + 30
121 -c 6
1400
128 f
133 + 31
1OOf 11
118 -c 15
103 + 12
>400
35
’ Group numbers correspond to those in Table 1. b Postgraft pancreatectomy. ’ No statistically significant differences between any of the experimental values and their corresponding control value (t test).
IVGTT curves (Fig. 1) and k values of 3.0 and 3.5, respectively, were nearly identical for both of these groups revealing a normal, rapid decay of glucose following intravenous glucose challenge. Four groups of rats were studied after receiving pancreata preserved by cold storage in
400
090
-
MIN WARM
0120
ii
MIN WARM
ISCHEMIA ISCHEMIA
2
i_: 300
h
0
~
5
10
15
20
1 60
30 TIME
(mid
FIG. 1. IVGTT (dextrose 500 mg/kg) curves for recipients of pancreatic grafts subjected to preharvest warm &hernia.
Collins’ solution. Eighty percent of the recipients receiving grafts stored for either 12 or 24 hr developed normoglycemia (groups 4 and 5, Table 1). Glucose values for the course of the experiment for these two successful groups are also listed in Table 2 and are not significantly different from those obtained in the warm ischemic or control animals either. The one failed animal in the 24-h preservation group had a blood glucose of 16 mg/dl on the first postoperative day and was found dead 1 day later which suggests that islet dysfunction with massive insulin release may have occurred. Likewise, the one failure in the 12-hr preservation group was found dead on the first postoperative day, possibly also having suffered from fatal hypoglycemia as an autopsy did not reveal any other obvious cause of death. Ability to handle an intravenous glucose challenge was not impaired by the hypothermic preservation for either 12 or 24 hr as indicated by normal IVGTT curves (Fig. 2) and k constants of 2.8 and 3.8 for the surviving animals in these two groups.
SCHULAK
AND
KISTHARD:
012
HOUR
COLD
ISCHEMIA
0 24 HOUR
COLD
ISCHEMIA
400
18
0
2
0
1.
5
10
15
PRESERVATION
1
20
60
30 TIME
hid
FIG. 2. IVGTT (dextrose 500 mg/kg) curves for recipients of pancreatic grafts subjected to pretransplant hypothermic preservation in Collins’ solution.
In contrast, extension of the period of coldstorage preservation past 24 hr was not well tolerated by the pancreatic islets in these experiments. Only two of the four recipients of pancreata stored for 36 hr became normoglycemic at 2 weeks, while neither of the two recipients of a 48-hr stored graft regained normal glucose homeostasis (groups 6 and 7, Table 1). The addition of preharvest warm ischemia to the insult of cold-hypothermic preservation reduced the amount of cold-storage time tolerated by these rat pancreatic grafts. One hour of warm ischemia prior to preservation was well tolerated by pancreata cold stored for only 12 hr. In this group (group 7, Table 1) six of seven recipients became normoglycemic following transplantation and had a mean blood glucose of 103 f 12 mg/dl at 2 weeks. Again, this value was not significantly different from either controls or the other successful experimental groups. In addition, response to glucose challenge for this group (group 7) was identical to that observed in the 12-hr preservation-alone group (group 3) as manifested both by very similar IVGTT curves (Figure 3) and k values of 3.0 and 2.8, respectively. Also, daily glucose values following transplantation were not remarkably different from those of the other successfulgroups (Table 2). On the other hand, 60 min of preharvest warm ischemia coupled with 24 hr of hypothermic
OF ISCHEMIC
PANCREAS
137
preservation resulted in nonfunction of all four grafts so attempted (group 8, Table 1). Neither increasing the warm ischemia time to 120 min and decreasingcold-storagetme to 6 hr (group 9, Table 1) nor decreasing warm time to 30 min and increasing preservation time to 18 hr (group 10, Table 1) was successful. All of these grafts either failed to reverse the pretransplant hyperglycemia or resulted in recipient death, again presumably due to hypoglycemia. Graft pancreatectomy was followed by a prompt return of hyperglycemia in all recipients of grafts with functioning islets suggesting that the level of glucose homeostasis observed was indeed due to the presence of the pancreatic graft. Histological review of these specimens from the normoglycemic recipients revealed abundant islets with normal morphology (Figure 4). On the other hand, islets were sparse, if present at all, in the grafts of the rats that were unsuccessfully transplanted suggesting that excessive ischemia whether cold or warm can result in islet degeneration. Acinar degeneration, fat necrosis, ductal dilatation, and varying degreesof inflammatory infiltration were observed in the exocrine pancreas of all specimens and thus cannot be solely attributed to ischemic injury as discussed previously [ 11.
500 .12
IIOUR
COLD
ISCHEMIA
4 0
5
10
15
20
30
60
TIME (mid
FIG. 3. IVGTI
(dextrose 500 mg/kg) curve for recipients of pancreatic grafts subjected to both preharvest warm ischemia and pretransplant hypothermic preservation in Collins’ solution compared to the IVGTT curve for recipients of nonischemic, cold-stored grafts.
138
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 36, NO. 2, FEBRUARY
1984
FIG. 4. Photomicrograph of a pancreatic graft subjected to I hr of preharvest warm &hernia and 12 hr of hypothermic preservation in Collins’ solution taken from a group 8 recipient with normal post-transplant glucose homeostasis. Note abundant, normal appearing islets (arrows) and the marked degeneration of surrounding acinar tissue (H&E, X80).
DISCUSSION
Successful hypothermic preservation of canine pancreatic grafts in iced intracellular electrolyte solution has been reported previously by others [4, 51. Whether simple cold storage is a more efficacious technique for pancreas preservation than pulsatile machine preservation is still controversial [6]. However, data recently reported by Florack et al. [7] comparing cold storage of canine pancreata in Collins’ solution, modified silica-gel-filtered plasma, and machine perfusion were clearly in favor of the nonperfusion technique. In none of these studies, however, was the question of whether a prior warm ischemic insult will adversely affect the ability to preserve the grafts by cold storage addressed. While Westbroek et al. [8] reported that 30 min of warm &hernia did not appreciably alter the outcome of machine perfused canine grafts they did
not study its effect on those that were cold stored. In clinical kidney transplantation, it is generally believed that warm ischemia shortens the safe period for subsequent graft preservation and thus it might be expected to have a similar influence on pancreas preservation and transplantation. In this study we achieved two goals. First, we demonstrated successful preservation and transplantation of rat pancreatic grafts using iced Collins’ solution as the storage medium. Normal glucose homeostasis was achieved immediately following transplantation in most recipients of organs stored for up to 24 hr. Longer periods of hypothermia were apparently harmful to the pancreata as only 50% of those hypothermically stored for 36 hr functioned and then somewhat sluggishly early on as indicated by their greater blood glucose values immediately following transplantation. Only two attempts at 48-hr storage were made
SCHULAK AND KISTHARD:
PRESERVATION OF ISCHEMIC PANCREAS
but both of these were clearly unsuccessful. Our observation of endocrine dysfunction with greater than 24 hr of cold storage is similar to that reported for canine pancreata also stored in Collins’ solution [5]. Nevertheless, this experiment demonstrates the feasibility of achieving successful nonperfusion hypothermic preservation of rat pancreata and thereby presents a model for subsequent study of this problem. The second and more important aim of this study was to evaluate the effect of preharvest warm ischemia on subsequent cold-storage preservation of the pancreas. Our data suggest that such an ischemic insult experienced prior to cooling may indeed significantly reduce the preservation potential for long-term hypothermic storage of this organ. While either 120 min or less of warm ischemia or up to 24 hr of cold storage were routinely well tolerated by the pancreatic grafts a combination of the two usually resulted in islet dysfunction. Only 12 hr of preservation were possible when warm ischemia was limited to 60 min or less. Lengthening the preservation time and decreasing the amount of warm ischemia did not permit adequate islet function. Similarly, decreasing storage time and increasing warm ischemia also resulted in failed grafts. These preliminary studies do not suggesta mechanism for the observations described. It may be speculated, however, that an ischemic insult of the hypothermic variety is additive to the warm induced injury of pancreatic islets that we described previously [ 11.Thus certain combinations of the two result in a cumulative injury that exceedsthe ability of the islets to recover and results in their rapid degeneration. In support of this hypothesis of additive injury is our recent observation that up to 5 hr of ambient temperature ischemia alone, as used in this study, was well tolerated by transplant
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pancreata in rats [9]. Lastly, whether or not hypothermic preservation of ischemic pancreatic grafts in media other than Collins’ solution may yield better results as has been suggestedfor kidneys [lo] is uncertain. Nevertheless, these experiments suggest that in clinical pancreas transplantation, an effort should be made to limit warm ischemic time to a minimum if the use of hypothermic preservation in Collins’ solution is contemplated. REFERENCES 1. Schulak, J. A., Franklin, W. A., Stuart, F. P., and Reckard, C. R. Effect of warm &hernia on segmental pancreas transplantation in the rat. Transplantation 35: 7, 1983. 2. Matas, A. J., Sutherland, D. E. R., Payne, W. D., Kretschmer, G. J., Steffes, M. W., and Najarian, J. S. The critical period of donor ischemia in neonatal rats. Transplantation 23: 295, 1977. 3. Williams, R. H., and Porte, D. The pancreas. In R. H. Williams (Ed.), Textbook of Endocrinology, Philadelphia: Saunders, 1974. P. 565. 4. Baumgartner, D., Sutherland, D. E. R., Heil, J. E., Zweber, B., Awad, E. A., and Najarian, J. S. Cold storage of segmental canine pancreatic grafts for 24 hours. J. Surg. Res. 29: 248, 1980. 5. Florack, G., Sutherland, D. E. R., Heil, J., Zweber, B., and Najarian, J. S. Long-term preservation of segmental pancreas autografts. Surgery 92: 260, 1982. 6. Toledo-Pereyra, L. H. Organ preservation. 1. Kidney and pancreas. J. Surg. Res. 30: 165, 1981. 7. Florack, G., Sutherland, D. E. R., Heil, J., Squifflet, J. P., and Najarian, J. S. Preservation of canine segmental pancreatic autografts: cold storage versus pulsame machine preservation. J. Surg. Res. 34: 493, 1983. 8. Westbroek, D. L., deGruy1, J., Digkhuis, C. M., et a/. Twenty-four-hour hypothermic preservation perfusion and storageof the duct-ligated canine pancreas with transplantation. Transplant Proc. 6: 319, 1974. 9. Schulak, J. A. Effect of post mortem ischemia on the function of adult rat islets following pancreatic transplantation. Metabolism 32: 643, 1983. 10. Halasz, N. A., and Collins, G. M. Forty-eight-hour kidney preservation. A comparison of flushing and ice storagewith perfusion. Arch. Surg. 111: 175, 1976.