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Continuous administration of pharmacological agents in heterotopically transplanted hearts
JOURNAL
OF SURGICAL
RESEARCH
41,473-478
(1986)
Continuous Administration of Pharmacological Agents in Heterotopically Transplanted Hearts R. W. F. DE BRUIN*,~-’ E. BOUWMAN, M.D.,? J. N. M. IJZERMANS, M.D.,? AND R. L. MARQUET, PH.D.*,? Department
of *Surgery, and tlaboratory Erasmus University, Rotterdam,
for Experimental The Netherlands
Surgery,
Submitted for publication August 20, 1985 A modification of the intraabdominal heterotopic heart transplantation model in rats is described which enables the local administration of pharmacological agents by means of a mini osmotic pump. In this modification, the aortic arch of the donor heart is cut after junction of the left carotid artery to enable the introduction of a catheter in the innominate artery. This catheter is connected to an implantable mini osmotic pump which delivers a continuous tlow for 7 days. Radiographs of transplanted animals bearing pumps filled with a contrast agent showed thatthe coronary vesselsof the graft were effectively perfused. Furthermore, continuous perfusion of prednisolone into the graft appeared to be as effective as subcutaneous infusion. Additional information about the usefulness of this perfusion technique is provided by recent data indicating that the expression of MHC class II antigens on vascular endothelium of a graft could only be. evoked by interferon-y when it was locally administered into the graft. Our results demonstrate that this technique is suitable for investigating the effects of local, continuous administration of pharmacological agents on heart grafts. 0 1986 Academic &SS Inc. INTRODUCTION
The heterotopic auxiliary heart transplantation technique in rats, first described by Abbot and Lindsey [I], is frequently used in transplantation studies. This model is less complicated than renal transplantation, mainly because there are no ureteric problems, and rejection is easily demonstrable. For several years this technique has been used by us to investigate the induction of specific and nonspecific immunosuppression [2-51. Recently, our attention has focused on the role of lymphokines in the rejection process [6]. Lymphokines are soluble substances produced by lymphocytes, which act as messengers in the immune system (e.g., interleukin-1, interleukin-2, and interferon-y). Lymphokines are rapidly inactivated in the organism and, therefore, local administration of these agents to the area where the rejection process is most prominent, namely the graft itself, may be ’ To whom request reprints should be addressed: Laboratory for Experimental Surgery, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 473
mandatory to produce any significant effect [7]. For this reason, the existing heterotopic heart transplantation technique was modified to enable the local administration of agents to the graft in a continuous way using an implantable osmotic pump. MATERIALS
AND
METHODS
Animals. Male rats of the inbred WAG (RT 1”) and BN strain (RTl”) were used. The animals were bred under specific pathogenfree conditions, were 12- 16 weeks old, and weighed 200-250 g. Osmotic pump. The pump (Alzet, Alza Corporation, Palo Alto, Calif.) acts by osmotic attraction of interstitial water in the outer layer. This results in pressure on the inner reservoir which is thereby compressed, releasing its contents at a constant flow rate. The pumps used in these experiments are the Alzet Model 2ML 1, with a reservoir volume of 2 ml and a nominal pumping rate of 10 &hr for 7 days. Heart transplantation. Essentially, the method consists of end to side anastomosis of 0022-4804/86 $1.50 Copyright 0 1986 by Academic Ress, Inc. All rights of reproduction in any form reserved.
474
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
the donor aorta with the recipient aorta, the donor pulmonary artery (p.a.) with the recipient inferior vena cava, introduction of a catheter in the innominate artery of the graft, and connection of the catheter to a mini osmotic pump. This provides the in vivo coronary perfusion of the graft both with recipient blood and with the pharmacological agent in the osmotic pump (Fig. 1). The operation is performed at a magnification of ~9 using a Zeiss operation microscope. The method is described in detail below. Donor procedure. The donor is anesthetized with ether. About 50 units of heparin (Thromboliquine, Organon, Oss, The Netherlands) is injected into the penile vein to prevent clotting in the heart. The thorax is opened via two lateral incisions and is retracted cranially. The thymus and pericardium are removed from the heart so that the aorta and p.a. become clearly visible. The aortic arch and its first two branches are carefully dissected from the surrounding tissue and from the p.a.. The p.a. is then cut with microvascular scissors just below the bifurcation. The aorta is cut after junction of the left carotid artery. The
FIG.
1986
pulmonary and caval veins are ligated with 2/O suture (Ethicon). The left carotid artery is ligated with 6/O suture, and the heart is perfused via the aorta with 4 ml of Hanks’ balanced salt solution (HBSS) to remove residual donor blood from the coronary system. The heart is stored in cold HBSS. Recipient procedure. The abdomen is opened and the intestines are moved to the left. The intraabdominal aorta and vena cava are dissected free and clamped together in a curved hemostat, just below the renal vessels. On the distal end, the vessels are occluded with a Schwartz clamp. Using microvascular scissors a hole, corresponding to the diameter of the aorta and p.a., is cut into the aorta and vena cava, respectively. Two stay sutures are placed through the angular points of the hole in the recipient aorta and the corresponding points of the donor aorta. The anastomosis is completed by continuous suturing of the vessel walls with 8/O monofil. The p.a. to vena cava anastomosis is accomplished in the same way with 7/O silk. The catheter (Silastic, outside diameter 1.1 mm) is filled with phosphatebuffered saline (PBS) containing 50 I.U. of
1. Modified heart transplantation. The catheter (arrow) is filled with PBS-heparin solution.
DE BRUIN
ET AL.: HEART
GRAFTS
475
heparin and is introduced in the innominate should have expelled about 0.9 ml of its conartery. It is tied firmly with two 6/O suture tents.) Once again, there was no detectable ligatures. The catheter is closed with a bulldog leakage (Fig. 4). Subsequently, the catheter was connected to a 2-ml syringe filled with the clamp to prevent blood loss via a retrograde flow through, and subsequent clotting in, the contrast agent. During visual observation on the X-ray monitor, the heart was perfused uncatheter. A dental cotton wool roll (Celluron) der low pressure. It could be clearly observed is placed on the anastomosis and the hemostatic clamps are released. The dental roll is that first the donor aorta filled with contrast gently pressed until hemorrhage is under con- agent, followed by the coronary vessels (Fig. trol. 5). Even at this relatively high pressure, as Connection and implantation of the pump. Once hemostasis is achieved, a hole is made in the mesocolon, the adjacent transverse abdominal muscle, and the overlying skin. The catheter is then passed under the colon and pulled through the hole in the muscle and skin. The catheter is fixed on the psoas muscle with a 7/O suture. The abdomen is closed in one layer with 2/O suture. A subcutaneous (s.c.) tunnel is made from an incision in the dorsal skin to the hole where the catheter leaves the abdomen. The catheter is pulled through this tunnel, and the mini osmotic pump is implanted S.C. in the interscapular area. The catheter is securely connected to the pump and is fixed on the S.C. fascia. The skin is closed with wound clips. Evaluation of graft function. Evaluation is done by daily palpation of the graft through the abdominal wall. The time at which regular heart beats can no longer be assessed is taken as the end point of graft survival. Testing of pump function. In order to determine the efficacy of this new technique, heart transplantation was performed as described. The pump was filled with 2 ml of the contrast agent meglumini iotalamas at a concentration of 600 mg/ml (Conray 60, Byk, The Netherlands). The catheter was filled with PBS-heparin solution. A radiograph was taken immediately after surgery (Fig. 2), the next X ray was taken 18 hr later. The catheter appeared to be filled with contrast agent, and no leakage could be detected either on the pump to catheter, or on the catheter to artery connection (Fig. 3). The X ray taken 4 days after surgery showed a marked decrease in reservoir FIG. 2. Radiograph taken directly after implantation of volume indicating that the pump did indeed the pump (dorsal view). The catheter is filled with PBSheparin solution and is hardly visible. function. (Theoretically, by now, the pump
476
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
1986
The pumps were removed after 7 days. The mean survivial time of the grafts was 15 * 3 days (Table 1, group 4). This is as effective as continuous subcutaneous infusion which gave a mean surival time of 15 f 5 days (group 3). There was no effect of the technical procedure itself on graft survival, as indicated by group 2. Survival times of this placebo-treated group were not different from those obtained in untreated control animals (group 1).
FIG. 3. Radiograph taken 18 hr after surgery (dorsal view). The catheter is filled with contrast agent. There is no detectable leakage at the catheter to pump, or at the catheter to artery connection.
compared to the pressure generated by the pump, there was no leakage at the catheter to artery connection. In addition, the efficacy of the modified heart transplantation technique was tested in the allogeneic BN to WAG combination. The osmotic pumps were filled with 15 mg prednisolone (Di- Adreson- F aquosum, Organon, Oss, The Netherlands), providing a daily dose of 8.5 mg/kg body wt for 7 days.
FIG. 4. Radiograph taken 4 days after surgery (lateral view). There is a diminution in pump volume indicating that the pump is operating normally. There is no detectable leakage.
DE BRUIN
ET AL.: HEART
477
GRAFIS
r-RIFN-y was administered in low and high doses, either systemic or locally into the grafts. The hearts were monitored for class II expression via an immunoperoxidase staining using anti-class II monoclonal antibodies. It was found that only grafts that were perfused with a high dose r-RIFN-y became class II positive [ 81. These results clearly indicate that the described perfusion technique is useful and effective. TECHNICAL
ANNOTATIONS
Apart from the technical problems that may occur during the procedure of normal heterotopic heart transplantation, such as bleeding at the site of the anastomosis, and constriction of the aorta, additional difficulties may be encountered in performing the modified technique.
FIG. 5. The donor heart is perfused under low pressure with contrast agent. The donor aorta (arrow l), and coronary vessels(arrow 2) are clearly visible.
An additional indication for the usefulness of the described method is provided by recent data from our laboratory concerning the administration of the lymphokine recombinant rat interferon-y (r-RIFN-y). The study was done to investigate whether r-RIFN-y had the capacity to induce the expression of MHC class II antigens on vascular endothelium. Briefly, syngeneic heart transplantation was performed according to the method described in this paper, or in the unmodified fashion.
(1) Improper preparation of the aortic arch may provide an innominate artery that is not suitable for the insertion of the catheter. Further, it is also important to cut the aortic arch distal of the left carotid artery, in order to obtain an aorta that has appropriate length to ensure that most of the agent expelled by the osmotic pump finds its way into the heart, and not into the aorta of the recipient. However, since there is also a retrogade flow in the donor aorta it is inevitable that a part of the pharmacological agent enters the systemic circulation first. (2) Dislodgement of the catheter from the innominate artery; therefore, it is imperative that the catheter is firmly fixed. (3) The catheter should be passed under the colon, if it is laid over the colon obstruction of the colon may occur. (4) The catheter should be filled with PBSheparin solution, and not with recipient blood in order to prevent clotting in the catheter. (5) The pump-as used in this papershould not be placed in the abdominal cavity because of its size; it may lead to necrosis of the intestines and to peritonitis. It is therefore mandatory to place the pump S.C.When using small pumps (e.g., the Alzet 2001 with a res-
478
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
1986
TABLE 1 SURVIVAL
TIMES
OF HEART
ALL~GRAFTS
IN RECIPIENTS
Treatment
n
1. No treatment 2. Continuous infusion of PBS in the graft 7 days 3. Continuous S.C.infusion of prednisolone, 8.5 mg/kg 4. Continuous infusion of prednisolone into the graft, 7 days, 8.5 mg/kg
25
TREATED
WITH
Survival time in days
PREDNISOLONE
Mean survival time + SD
8-9
8.5 + 0.5
4
8, 8, 8, 10
8.5 f 1
4
10, 13, 17, 21
15 +5
5
11, 13, 14, 18, 18
15 +3
ervoir volume of 0.2 ml), s.c. implantation is not necessary. The pump can then be placed intraabdominal, and be anchored under the peritoneum. ACKNOWLEDGMENTS We thank Mrs. W. van Leeuwen for preparing the radiographs and Dr. B. Tank for his careful editing of the English text. REFERENCES 1. Abbott, C. P., and Lindsey, E. S. A technique for heart transplantation in the rat. Arch. Surg. 89: 645, 1964. 2. Marquet, R. L., Heystek, G. A., Cobussen, A. C., Niessen, G. J. C. M., and Jeekel, J. Cyclosporin A prolongs graft survival in presensitized animals. Transpl. Pm. 15: 518, 1983. 3. Marquet, R. L., Heystek, G. A., Niessen, G. J. C. M., and Jeekel, J. Induction of suppressor cells by a single blood transfusion in rats. Trunspl. Pm. 14: 397, 1982.
4. Heineman, E., Marquet, R. L., Heystek, G. A., Cobussen, A., and Jeekel, J. Modification of allogratt rejection in rats by blood transfusions to the donor. Transpl. Proc. 14: 994, 1983. 5. Heineman, E., Bouwman, E., de Bruin, R. W. F., Marquet, R. L., and Jeekel, J. The role of passenger leucocytes in the manifestation of the DST phenomenon. Transpl. Proc. 17: 821, 1985. 6. Ijzermans, J. N. M., de Bruin, R. W. F., Schellekens, H., v.d. Meide, P. H., Weimar, W., and Marquet, R. L. Effects of low and high doses interferon on renal graft survival in rats. Trunspl. Proc. 17: 1409, 1985. 7. Ijzermans, J. N. M., Bijma, A. M., v.d. Meide, P. H., Schellekens, H., and Marquet, R. L. Antitumor and immunomodulating effectsof recombinant interlerony in rats. In H. Kirchner and H. Schellekens (Ed.), The Biology of the Inte&ron System 1984. New York: Elsevier 1985. P. 475. 8. Ijzermans, J. N. M., Marquet, R. L., Bouwman, E., de Bruin, R. W. F., Schellekens, H., and Jeekel, J. In vivo induction of class II antigens by rat recombinant interferon-y. In Stewart and Schellekens (Ed.), The Biology of the Interferon System 1985. New York: Elsevier 1986. P. 355-358.
OF SURGICAL
RESEARCH
41,473-478
(1986)
Continuous Administration of Pharmacological Agents in Heterotopically Transplanted Hearts R. W. F. DE BRUIN*,~-’ E. BOUWMAN, M.D.,? J. N. M. IJZERMANS, M.D.,? AND R. L. MARQUET, PH.D.*,? Department
of *Surgery, and tlaboratory Erasmus University, Rotterdam,
for Experimental The Netherlands
Surgery,
Submitted for publication August 20, 1985 A modification of the intraabdominal heterotopic heart transplantation model in rats is described which enables the local administration of pharmacological agents by means of a mini osmotic pump. In this modification, the aortic arch of the donor heart is cut after junction of the left carotid artery to enable the introduction of a catheter in the innominate artery. This catheter is connected to an implantable mini osmotic pump which delivers a continuous tlow for 7 days. Radiographs of transplanted animals bearing pumps filled with a contrast agent showed thatthe coronary vesselsof the graft were effectively perfused. Furthermore, continuous perfusion of prednisolone into the graft appeared to be as effective as subcutaneous infusion. Additional information about the usefulness of this perfusion technique is provided by recent data indicating that the expression of MHC class II antigens on vascular endothelium of a graft could only be. evoked by interferon-y when it was locally administered into the graft. Our results demonstrate that this technique is suitable for investigating the effects of local, continuous administration of pharmacological agents on heart grafts. 0 1986 Academic &SS Inc. INTRODUCTION
The heterotopic auxiliary heart transplantation technique in rats, first described by Abbot and Lindsey [I], is frequently used in transplantation studies. This model is less complicated than renal transplantation, mainly because there are no ureteric problems, and rejection is easily demonstrable. For several years this technique has been used by us to investigate the induction of specific and nonspecific immunosuppression [2-51. Recently, our attention has focused on the role of lymphokines in the rejection process [6]. Lymphokines are soluble substances produced by lymphocytes, which act as messengers in the immune system (e.g., interleukin-1, interleukin-2, and interferon-y). Lymphokines are rapidly inactivated in the organism and, therefore, local administration of these agents to the area where the rejection process is most prominent, namely the graft itself, may be ’ To whom request reprints should be addressed: Laboratory for Experimental Surgery, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. 473
mandatory to produce any significant effect [7]. For this reason, the existing heterotopic heart transplantation technique was modified to enable the local administration of agents to the graft in a continuous way using an implantable osmotic pump. MATERIALS
AND
METHODS
Animals. Male rats of the inbred WAG (RT 1”) and BN strain (RTl”) were used. The animals were bred under specific pathogenfree conditions, were 12- 16 weeks old, and weighed 200-250 g. Osmotic pump. The pump (Alzet, Alza Corporation, Palo Alto, Calif.) acts by osmotic attraction of interstitial water in the outer layer. This results in pressure on the inner reservoir which is thereby compressed, releasing its contents at a constant flow rate. The pumps used in these experiments are the Alzet Model 2ML 1, with a reservoir volume of 2 ml and a nominal pumping rate of 10 &hr for 7 days. Heart transplantation. Essentially, the method consists of end to side anastomosis of 0022-4804/86 $1.50 Copyright 0 1986 by Academic Ress, Inc. All rights of reproduction in any form reserved.
474
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
the donor aorta with the recipient aorta, the donor pulmonary artery (p.a.) with the recipient inferior vena cava, introduction of a catheter in the innominate artery of the graft, and connection of the catheter to a mini osmotic pump. This provides the in vivo coronary perfusion of the graft both with recipient blood and with the pharmacological agent in the osmotic pump (Fig. 1). The operation is performed at a magnification of ~9 using a Zeiss operation microscope. The method is described in detail below. Donor procedure. The donor is anesthetized with ether. About 50 units of heparin (Thromboliquine, Organon, Oss, The Netherlands) is injected into the penile vein to prevent clotting in the heart. The thorax is opened via two lateral incisions and is retracted cranially. The thymus and pericardium are removed from the heart so that the aorta and p.a. become clearly visible. The aortic arch and its first two branches are carefully dissected from the surrounding tissue and from the p.a.. The p.a. is then cut with microvascular scissors just below the bifurcation. The aorta is cut after junction of the left carotid artery. The
FIG.
1986
pulmonary and caval veins are ligated with 2/O suture (Ethicon). The left carotid artery is ligated with 6/O suture, and the heart is perfused via the aorta with 4 ml of Hanks’ balanced salt solution (HBSS) to remove residual donor blood from the coronary system. The heart is stored in cold HBSS. Recipient procedure. The abdomen is opened and the intestines are moved to the left. The intraabdominal aorta and vena cava are dissected free and clamped together in a curved hemostat, just below the renal vessels. On the distal end, the vessels are occluded with a Schwartz clamp. Using microvascular scissors a hole, corresponding to the diameter of the aorta and p.a., is cut into the aorta and vena cava, respectively. Two stay sutures are placed through the angular points of the hole in the recipient aorta and the corresponding points of the donor aorta. The anastomosis is completed by continuous suturing of the vessel walls with 8/O monofil. The p.a. to vena cava anastomosis is accomplished in the same way with 7/O silk. The catheter (Silastic, outside diameter 1.1 mm) is filled with phosphatebuffered saline (PBS) containing 50 I.U. of
1. Modified heart transplantation. The catheter (arrow) is filled with PBS-heparin solution.
DE BRUIN
ET AL.: HEART
GRAFTS
475
heparin and is introduced in the innominate should have expelled about 0.9 ml of its conartery. It is tied firmly with two 6/O suture tents.) Once again, there was no detectable ligatures. The catheter is closed with a bulldog leakage (Fig. 4). Subsequently, the catheter was connected to a 2-ml syringe filled with the clamp to prevent blood loss via a retrograde flow through, and subsequent clotting in, the contrast agent. During visual observation on the X-ray monitor, the heart was perfused uncatheter. A dental cotton wool roll (Celluron) der low pressure. It could be clearly observed is placed on the anastomosis and the hemostatic clamps are released. The dental roll is that first the donor aorta filled with contrast gently pressed until hemorrhage is under con- agent, followed by the coronary vessels (Fig. trol. 5). Even at this relatively high pressure, as Connection and implantation of the pump. Once hemostasis is achieved, a hole is made in the mesocolon, the adjacent transverse abdominal muscle, and the overlying skin. The catheter is then passed under the colon and pulled through the hole in the muscle and skin. The catheter is fixed on the psoas muscle with a 7/O suture. The abdomen is closed in one layer with 2/O suture. A subcutaneous (s.c.) tunnel is made from an incision in the dorsal skin to the hole where the catheter leaves the abdomen. The catheter is pulled through this tunnel, and the mini osmotic pump is implanted S.C. in the interscapular area. The catheter is securely connected to the pump and is fixed on the S.C. fascia. The skin is closed with wound clips. Evaluation of graft function. Evaluation is done by daily palpation of the graft through the abdominal wall. The time at which regular heart beats can no longer be assessed is taken as the end point of graft survival. Testing of pump function. In order to determine the efficacy of this new technique, heart transplantation was performed as described. The pump was filled with 2 ml of the contrast agent meglumini iotalamas at a concentration of 600 mg/ml (Conray 60, Byk, The Netherlands). The catheter was filled with PBS-heparin solution. A radiograph was taken immediately after surgery (Fig. 2), the next X ray was taken 18 hr later. The catheter appeared to be filled with contrast agent, and no leakage could be detected either on the pump to catheter, or on the catheter to artery connection (Fig. 3). The X ray taken 4 days after surgery showed a marked decrease in reservoir FIG. 2. Radiograph taken directly after implantation of volume indicating that the pump did indeed the pump (dorsal view). The catheter is filled with PBSheparin solution and is hardly visible. function. (Theoretically, by now, the pump
476
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
1986
The pumps were removed after 7 days. The mean survivial time of the grafts was 15 * 3 days (Table 1, group 4). This is as effective as continuous subcutaneous infusion which gave a mean surival time of 15 f 5 days (group 3). There was no effect of the technical procedure itself on graft survival, as indicated by group 2. Survival times of this placebo-treated group were not different from those obtained in untreated control animals (group 1).
FIG. 3. Radiograph taken 18 hr after surgery (dorsal view). The catheter is filled with contrast agent. There is no detectable leakage at the catheter to pump, or at the catheter to artery connection.
compared to the pressure generated by the pump, there was no leakage at the catheter to artery connection. In addition, the efficacy of the modified heart transplantation technique was tested in the allogeneic BN to WAG combination. The osmotic pumps were filled with 15 mg prednisolone (Di- Adreson- F aquosum, Organon, Oss, The Netherlands), providing a daily dose of 8.5 mg/kg body wt for 7 days.
FIG. 4. Radiograph taken 4 days after surgery (lateral view). There is a diminution in pump volume indicating that the pump is operating normally. There is no detectable leakage.
DE BRUIN
ET AL.: HEART
477
GRAFIS
r-RIFN-y was administered in low and high doses, either systemic or locally into the grafts. The hearts were monitored for class II expression via an immunoperoxidase staining using anti-class II monoclonal antibodies. It was found that only grafts that were perfused with a high dose r-RIFN-y became class II positive [ 81. These results clearly indicate that the described perfusion technique is useful and effective. TECHNICAL
ANNOTATIONS
Apart from the technical problems that may occur during the procedure of normal heterotopic heart transplantation, such as bleeding at the site of the anastomosis, and constriction of the aorta, additional difficulties may be encountered in performing the modified technique.
FIG. 5. The donor heart is perfused under low pressure with contrast agent. The donor aorta (arrow l), and coronary vessels(arrow 2) are clearly visible.
An additional indication for the usefulness of the described method is provided by recent data from our laboratory concerning the administration of the lymphokine recombinant rat interferon-y (r-RIFN-y). The study was done to investigate whether r-RIFN-y had the capacity to induce the expression of MHC class II antigens on vascular endothelium. Briefly, syngeneic heart transplantation was performed according to the method described in this paper, or in the unmodified fashion.
(1) Improper preparation of the aortic arch may provide an innominate artery that is not suitable for the insertion of the catheter. Further, it is also important to cut the aortic arch distal of the left carotid artery, in order to obtain an aorta that has appropriate length to ensure that most of the agent expelled by the osmotic pump finds its way into the heart, and not into the aorta of the recipient. However, since there is also a retrogade flow in the donor aorta it is inevitable that a part of the pharmacological agent enters the systemic circulation first. (2) Dislodgement of the catheter from the innominate artery; therefore, it is imperative that the catheter is firmly fixed. (3) The catheter should be passed under the colon, if it is laid over the colon obstruction of the colon may occur. (4) The catheter should be filled with PBSheparin solution, and not with recipient blood in order to prevent clotting in the catheter. (5) The pump-as used in this papershould not be placed in the abdominal cavity because of its size; it may lead to necrosis of the intestines and to peritonitis. It is therefore mandatory to place the pump S.C.When using small pumps (e.g., the Alzet 2001 with a res-
478
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 5, NOVEMBER
1986
TABLE 1 SURVIVAL
TIMES
OF HEART
ALL~GRAFTS
IN RECIPIENTS
Treatment
n
1. No treatment 2. Continuous infusion of PBS in the graft 7 days 3. Continuous S.C.infusion of prednisolone, 8.5 mg/kg 4. Continuous infusion of prednisolone into the graft, 7 days, 8.5 mg/kg
25
TREATED
WITH
Survival time in days
PREDNISOLONE
Mean survival time + SD
8-9
8.5 + 0.5
4
8, 8, 8, 10
8.5 f 1
4
10, 13, 17, 21
15 +5
5
11, 13, 14, 18, 18
15 +3
ervoir volume of 0.2 ml), s.c. implantation is not necessary. The pump can then be placed intraabdominal, and be anchored under the peritoneum. ACKNOWLEDGMENTS We thank Mrs. W. van Leeuwen for preparing the radiographs and Dr. B. Tank for his careful editing of the English text. REFERENCES 1. Abbott, C. P., and Lindsey, E. S. A technique for heart transplantation in the rat. Arch. Surg. 89: 645, 1964. 2. Marquet, R. L., Heystek, G. A., Cobussen, A. C., Niessen, G. J. C. M., and Jeekel, J. Cyclosporin A prolongs graft survival in presensitized animals. Transpl. Pm. 15: 518, 1983. 3. Marquet, R. L., Heystek, G. A., Niessen, G. J. C. M., and Jeekel, J. Induction of suppressor cells by a single blood transfusion in rats. Trunspl. Pm. 14: 397, 1982.
4. Heineman, E., Marquet, R. L., Heystek, G. A., Cobussen, A., and Jeekel, J. Modification of allogratt rejection in rats by blood transfusions to the donor. Transpl. Proc. 14: 994, 1983. 5. Heineman, E., Bouwman, E., de Bruin, R. W. F., Marquet, R. L., and Jeekel, J. The role of passenger leucocytes in the manifestation of the DST phenomenon. Transpl. Proc. 17: 821, 1985. 6. Ijzermans, J. N. M., de Bruin, R. W. F., Schellekens, H., v.d. Meide, P. H., Weimar, W., and Marquet, R. L. Effects of low and high doses interferon on renal graft survival in rats. Trunspl. Proc. 17: 1409, 1985. 7. Ijzermans, J. N. M., Bijma, A. M., v.d. Meide, P. H., Schellekens, H., and Marquet, R. L. Antitumor and immunomodulating effectsof recombinant interlerony in rats. In H. Kirchner and H. Schellekens (Ed.), The Biology of the Inte&ron System 1984. New York: Elsevier 1985. P. 475. 8. Ijzermans, J. N. M., Marquet, R. L., Bouwman, E., de Bruin, R. W. F., Schellekens, H., and Jeekel, J. In vivo induction of class II antigens by rat recombinant interferon-y. In Stewart and Schellekens (Ed.), The Biology of the Interferon System 1985. New York: Elsevier 1986. P. 355-358.