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Pig to Canine Auxiliary Hepatic Xenotransplantation Model: Prevention of Hyperacute Rejection Via Kupffer Cell Blockade and Complement Regulation
XENOGRAFTS
Pig to Canine Auxiliary Hepatic Xenotransplantation Model: Prevention of Hyperacute Rejection Via Kupffer Cell Blockade and Complement Regulation K.-Y. Chung, J.-J. Park, and K.-H. Han ABSTRACT Objectives. Large animal experiment models are a critical prerequisite to preclinical trials. However, the pig-to-primate model is expensive and proper experimental conditions are difficult to establish. Several pig-to-canine lung xenotransplantation experiments have shown hyperacute rejection. Therefore, we designed a pig-to-canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences after xenotransplantation and hyperacute rejection. Methods. Animals were divided into two groups of 3 each: a cobra venom factor plus gadolinium trichloride (GdCl3) treatment group (CVF⫹Gd group) and a control group. Whole livers from 15-kg donor pigs were harvested and perfused with histidine-tryptophanketoglutarate solution. Seventy percent of the left lobe of the livers of 17-kg recipient dogs was resected. The harvested pig whole liver was transplanted using the canine left hepatic vein, left portal vein, and common hepatic artery. After graft reperfusion, blood samples and aliquots of liver, lung, and kidney tissues were obtained at 1 hour after reperfusion. Results. We successfully completed 6 pig-to-canine auxiliary hepatic xenotransplantations. In the control group, the grafts showed a patchy hypoperfused liver surface that was rubbery solid compared with the CVF⫹Gd group. Serum total protein, albumin, fibrinogen, and platelet counts decreased abruptly; however, there were no significant differences between the two groups. There were no identifiable changes in blood urea nitrogen and creatinine concentrations. Serum prothrombin time, partial thromboplastin time, and further degradation product were increased in both groups; however, in the CVF⫹Gd group, the slope was more obtuse than in the control group. At microscopy, the graft at 20, 40, and 60 minutes after reperfusion, no intravascular pathologic changes were noted. Only scant intravascular fibrin
From the Departments of Surgery (K.-Y.C., J.-J.P.) and Anatomy (K.-H.H.), Ewha Womans University School of Medicine and Ewha Medical Research Center (K.-Y.C., K.-H.H.), Seoul, Korea. This study was supported by (grant KRF-2005-042-E00075) from the Korea Research Foundation.
Address reprint requests to K.Y. Chung, MD, Department of Surgery, Ewha Womans University Hospital, Mok 6 Dong 911-1 Yang-Cheon-Ku, Seoul 159-710, South Korea. E-mail: kuyong@ ewha.ac.kr
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.056
Transplantation Proceedings, 40, 2755–2759 (2008)
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deposition was observed. Hepatocellular vacuolization and sinusoidal dilatation were seen. There was patch necrosis without a zonal distribution, and intrasinusoidal neutrophil sequestration and interstitial hemorrhage. These findings were milder in the CVF⫹Gd group. Conclusion. A pig-to-canine partial auxiliary liver xenotransplantation model is feasible. In the CVF⫹Gd treatment group, pathologic findings of patch hepatocyte necrosis were less severe. Inasmuch there was no corresponding vascular pathologic finding, these abnormalities are not a direct effect of CVF⫹Gd treatment. Other factors such as ischemia-reperfusion injury should be considered.
A
lthough concordant xenotransplantation using a primate organ is more physiologic and is assumed to be better to overcome immunologic barriers, the pig is suitable for performance of genetic modifications, is easy to breed, and grows rapidly, and there are fewer ethical issues than with primate organs.1 Gal (alpha 1,3)Gal antigen and inadequate complement regulation is a major hurdle to xenotransplantation leading to hyperacute rejection, which is to be overcome with genetic modification of pigs.2,3 The next obstacle to hyperacute rejection is acute humoral xenograft rejection.4 To manage these problems, a large animal experiment model is a critical prerequisite to a preclinical trial. However, the pig-toprimate model is expensive and experimental conditions are difficult to establish. Although the pig-to-canine model is different from the pig-to-primate model, several pig-to-canine lung xenotransplantation experiments have revealed hyperacute rejection.5 Therefore, we designed a pig-to-canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences after xenotransplantation and hyperacute rejection. MATERIALS AND METHODS Mongrel dogs of both sexes weighing 15 to 25 kg and 3-month-old pigs weighing 15 to 18 kg were used after being fasted for 12 hours. Animals were treated in compliance with the guidelines of our animal research committee. Isoflurane (Forane; ChoongWei Pharma Co Ltd, Seoul, Korea) was used for anesthetic maintenance with intermittent pancuronium bromide (Panslane; Reyon Pharmacutical Co Ltd, Seoul Korea) injections for muscle relaxation. Animals were divided into two groups: of three each: a treatment group and a control. On day 1, the treatment group received cobra venom factor, 50 g/kg IV, plus gadolinium trichloride (GdCl3), 20 mg/kg; the control group received none of these premedications.
Donor Hepatectomy The donor pig abdomen was opened via a midline incision. After intravenous infusion of 5000 U of heparin sodium, the common hepatic artery and portal vein were intubated and perfused with histidine-tryptophan-ketoglutarate solution. The entire liver was harvested and stored in a refrigerator.
Auxiliary Hepatic Xenotransplantation The recipient canine abdomen was opened via a chevron incision extending to the xiphoid process. The left hepatic duct was divided at just above the bifurcation. The gastroduodenal artery was ligated and
divided. The common hepatic artery was looped, at the branch of the right hepatic artery. The gastroduodenal artery was ligated and divided. After exposing the anterior aspect of the portal bifurcation, two or three fine portal branches to the caudate segment were double ligated and cut to prepare the left portal vein about 2 cm length. The middle hepatic vein was isolated and divided after distal ligation and proximal suture ligation with 5.0 polypropalene sutures. The left hepatic vein was isolated and looped with a rubberband for later clamping. Then the left partial graft including the right medial, quadrate, left medial, left lateral segment, and the papillary process of the caudate lobe were resected. The stored whole pig liver was transplanted using the canine left hepatic vein, left portal vein, and common hepatic artery. A blood sample and a liver biopsy specimen were obtained just before partial hepatectomy and postreperfusion at 20, 40, and 60 minutes.
RESULTS
We successfully performed all 6 pig-to-canine auxiliary hepatic xenotransplantations. During reperfusion, the blood pressure dropped acutely; however, after infusion of saline solution, the vital signs stabilized by 1 hour after reperfusion. In the control group, the grafts showed a patchy hypoperfused surface and felt rubbery and solid compared with the CVF⫹Gd group (Fig 1). The Serum total protein and albumin levels decreased abruptly; however, there were no significant differences between the two groups (Fig 2). There was no identifiable change in blood urea nitrogen or creatinine levels (Fig 2). The Serum fibrinogen level decreased sharply, and the CVF⫹Gd group showed a more obtuse slope than the control group (Fig 3). Fibrinogen degradation product increased in both groups with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 3). The platelet counts decreased in both groups with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 2). Serum prothrombin time and partial thromboplastin time increased in both groups, with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 3). At microscopic examination of the graft at 20, 40, and 60 minutes after reperfusion, we did not observe severe intravascular coagulation, and there was only scant intravascular fibrin deposition. Hepatocellular vacuolization and sinusoidal dilatation were accompanied by patch necrosis without zonal distribution, intrasinusoidal neutrophil sequestration, or interstitial hemorrhage. These findings were milder in the CVF⫹Gd group (Fig 4).
PIG TO CANINE HEPATIC XENOTRANSPLANTATION MODEL
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Fig 1. (A) Harvested donor pig liver. Views after canine partial hepatectomy (B); vascular anastomosis, portal vein side (C); vasdcular anastomosis, hepatic vein side (D); and reperfusion in the complement and Kupffer cell regulation group (E) and the control group (F). CBD, common bile duct; CHA, common hepatic artery; CHD, common hepatic duct; HV, left hepatic vein; IHVC, infrahepatic vena cava; LPV, left portal vein; PV, portal vein; Remnant RL, remnant canine right liver lobe segment; RPV, right portal vein.
Fig 2. Serum laboratory values after reperfusion. CVF, cobra venom factor.
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Fig 3. Serum values after reperfusion. aPTT, activated partial thromboplastin time; FDP, fibrinogen degradation product; PT, prothrombin time.
Fig 4. (A) Healthy pig liver after harvest. (B) Xenograft 60 minutes after reperfusion in the control group. (C) Xenograft 60 minutes after reperfusion in the treatment group. (D) Remnant dog liver 60 minutes after reperfusion.
PIG TO CANINE HEPATIC XENOTRANSPLANTATION MODEL
DISCUSSION
The Pig-to-canine partial auxiliary liver xenotransplantation model is feasible. The donor pig body weight was about 5 kg less than the recipient. The pig suprahepatic vena cava was about 2.2 cm in greatest diameter, and the canine left hepatic vein was 2 cm, which was widened with oblique incision. The pig portal vein diameter was approximately 1.8 cm, and the canine left portal vein was about 1.3 cm, which was also widened with oblique incision. There were rapid intravascular biochemical changes which is reflecting hyperacute rejection. However, there was no severe aberrant wound bleeding or anastomosis site bleeding. There was no microscopic intravascular coagulation suggestive of hyperacute rejection. Although intravascular biochemical changes were less severe in the CVF⫹Gd treatment group, more cases are needed to obtain statistical significance. In the CVF⫹Gd treatment group, the pathologic findings (eg, patch hepatocyte necrosis) were less severe. Inasmuch as there were no corresponding vascular pathologic findings, these findings could
2759
not be a direct effect of CVF⫹Gd treatment. Other factors such as ischemia-reperfusion injury should be considered to explain these finding. REFERENCES 1. Byrne G, McCurry K, Martin M, et al: Transgenic pigs expressing human CD59 and decay accelerating factor produce an intrinsic barrier to complement-mediated damage. Transplantation 63:149, 1997 2. Cozzi E, Tucker AW, Langford GA, et al: Characterization of pigs transgenic for human decay- accelerating factor. Transplantation 64:1383, 1997 3. Lin SS, Hanaway MJ, Gonzalez SG, et al: The role of anti-Gal(alpha1–3)Gal antibodies in acute vascular rejection and accommodation of xenografts. Transplantation 70:1667, 2000 4. Loss M, Vangerow B, Schmidtko J, et al: Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model. Xenotransplantation 7:186, 2000 5. Nakajima R, Nakajima S, Nagata S, et al: Analysis of hyperacute rejection in newborn pig to dog lung xenotransplantation. Transplant Proc 32:1131, 2000
Pig to Canine Auxiliary Hepatic Xenotransplantation Model: Prevention of Hyperacute Rejection Via Kupffer Cell Blockade and Complement Regulation K.-Y. Chung, J.-J. Park, and K.-H. Han ABSTRACT Objectives. Large animal experiment models are a critical prerequisite to preclinical trials. However, the pig-to-primate model is expensive and proper experimental conditions are difficult to establish. Several pig-to-canine lung xenotransplantation experiments have shown hyperacute rejection. Therefore, we designed a pig-to-canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences after xenotransplantation and hyperacute rejection. Methods. Animals were divided into two groups of 3 each: a cobra venom factor plus gadolinium trichloride (GdCl3) treatment group (CVF⫹Gd group) and a control group. Whole livers from 15-kg donor pigs were harvested and perfused with histidine-tryptophanketoglutarate solution. Seventy percent of the left lobe of the livers of 17-kg recipient dogs was resected. The harvested pig whole liver was transplanted using the canine left hepatic vein, left portal vein, and common hepatic artery. After graft reperfusion, blood samples and aliquots of liver, lung, and kidney tissues were obtained at 1 hour after reperfusion. Results. We successfully completed 6 pig-to-canine auxiliary hepatic xenotransplantations. In the control group, the grafts showed a patchy hypoperfused liver surface that was rubbery solid compared with the CVF⫹Gd group. Serum total protein, albumin, fibrinogen, and platelet counts decreased abruptly; however, there were no significant differences between the two groups. There were no identifiable changes in blood urea nitrogen and creatinine concentrations. Serum prothrombin time, partial thromboplastin time, and further degradation product were increased in both groups; however, in the CVF⫹Gd group, the slope was more obtuse than in the control group. At microscopy, the graft at 20, 40, and 60 minutes after reperfusion, no intravascular pathologic changes were noted. Only scant intravascular fibrin
From the Departments of Surgery (K.-Y.C., J.-J.P.) and Anatomy (K.-H.H.), Ewha Womans University School of Medicine and Ewha Medical Research Center (K.-Y.C., K.-H.H.), Seoul, Korea. This study was supported by (grant KRF-2005-042-E00075) from the Korea Research Foundation.
Address reprint requests to K.Y. Chung, MD, Department of Surgery, Ewha Womans University Hospital, Mok 6 Dong 911-1 Yang-Cheon-Ku, Seoul 159-710, South Korea. E-mail: kuyong@ ewha.ac.kr
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.056
Transplantation Proceedings, 40, 2755–2759 (2008)
2755
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deposition was observed. Hepatocellular vacuolization and sinusoidal dilatation were seen. There was patch necrosis without a zonal distribution, and intrasinusoidal neutrophil sequestration and interstitial hemorrhage. These findings were milder in the CVF⫹Gd group. Conclusion. A pig-to-canine partial auxiliary liver xenotransplantation model is feasible. In the CVF⫹Gd treatment group, pathologic findings of patch hepatocyte necrosis were less severe. Inasmuch there was no corresponding vascular pathologic finding, these abnormalities are not a direct effect of CVF⫹Gd treatment. Other factors such as ischemia-reperfusion injury should be considered.
A
lthough concordant xenotransplantation using a primate organ is more physiologic and is assumed to be better to overcome immunologic barriers, the pig is suitable for performance of genetic modifications, is easy to breed, and grows rapidly, and there are fewer ethical issues than with primate organs.1 Gal (alpha 1,3)Gal antigen and inadequate complement regulation is a major hurdle to xenotransplantation leading to hyperacute rejection, which is to be overcome with genetic modification of pigs.2,3 The next obstacle to hyperacute rejection is acute humoral xenograft rejection.4 To manage these problems, a large animal experiment model is a critical prerequisite to a preclinical trial. However, the pig-toprimate model is expensive and experimental conditions are difficult to establish. Although the pig-to-canine model is different from the pig-to-primate model, several pig-to-canine lung xenotransplantation experiments have revealed hyperacute rejection.5 Therefore, we designed a pig-to-canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences after xenotransplantation and hyperacute rejection. MATERIALS AND METHODS Mongrel dogs of both sexes weighing 15 to 25 kg and 3-month-old pigs weighing 15 to 18 kg were used after being fasted for 12 hours. Animals were treated in compliance with the guidelines of our animal research committee. Isoflurane (Forane; ChoongWei Pharma Co Ltd, Seoul, Korea) was used for anesthetic maintenance with intermittent pancuronium bromide (Panslane; Reyon Pharmacutical Co Ltd, Seoul Korea) injections for muscle relaxation. Animals were divided into two groups: of three each: a treatment group and a control. On day 1, the treatment group received cobra venom factor, 50 g/kg IV, plus gadolinium trichloride (GdCl3), 20 mg/kg; the control group received none of these premedications.
Donor Hepatectomy The donor pig abdomen was opened via a midline incision. After intravenous infusion of 5000 U of heparin sodium, the common hepatic artery and portal vein were intubated and perfused with histidine-tryptophan-ketoglutarate solution. The entire liver was harvested and stored in a refrigerator.
Auxiliary Hepatic Xenotransplantation The recipient canine abdomen was opened via a chevron incision extending to the xiphoid process. The left hepatic duct was divided at just above the bifurcation. The gastroduodenal artery was ligated and
divided. The common hepatic artery was looped, at the branch of the right hepatic artery. The gastroduodenal artery was ligated and divided. After exposing the anterior aspect of the portal bifurcation, two or three fine portal branches to the caudate segment were double ligated and cut to prepare the left portal vein about 2 cm length. The middle hepatic vein was isolated and divided after distal ligation and proximal suture ligation with 5.0 polypropalene sutures. The left hepatic vein was isolated and looped with a rubberband for later clamping. Then the left partial graft including the right medial, quadrate, left medial, left lateral segment, and the papillary process of the caudate lobe were resected. The stored whole pig liver was transplanted using the canine left hepatic vein, left portal vein, and common hepatic artery. A blood sample and a liver biopsy specimen were obtained just before partial hepatectomy and postreperfusion at 20, 40, and 60 minutes.
RESULTS
We successfully performed all 6 pig-to-canine auxiliary hepatic xenotransplantations. During reperfusion, the blood pressure dropped acutely; however, after infusion of saline solution, the vital signs stabilized by 1 hour after reperfusion. In the control group, the grafts showed a patchy hypoperfused surface and felt rubbery and solid compared with the CVF⫹Gd group (Fig 1). The Serum total protein and albumin levels decreased abruptly; however, there were no significant differences between the two groups (Fig 2). There was no identifiable change in blood urea nitrogen or creatinine levels (Fig 2). The Serum fibrinogen level decreased sharply, and the CVF⫹Gd group showed a more obtuse slope than the control group (Fig 3). Fibrinogen degradation product increased in both groups with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 3). The platelet counts decreased in both groups with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 2). Serum prothrombin time and partial thromboplastin time increased in both groups, with the CVF⫹Gd group showing a more obtuse slope than the control group (Fig 3). At microscopic examination of the graft at 20, 40, and 60 minutes after reperfusion, we did not observe severe intravascular coagulation, and there was only scant intravascular fibrin deposition. Hepatocellular vacuolization and sinusoidal dilatation were accompanied by patch necrosis without zonal distribution, intrasinusoidal neutrophil sequestration, or interstitial hemorrhage. These findings were milder in the CVF⫹Gd group (Fig 4).
PIG TO CANINE HEPATIC XENOTRANSPLANTATION MODEL
2757
Fig 1. (A) Harvested donor pig liver. Views after canine partial hepatectomy (B); vascular anastomosis, portal vein side (C); vasdcular anastomosis, hepatic vein side (D); and reperfusion in the complement and Kupffer cell regulation group (E) and the control group (F). CBD, common bile duct; CHA, common hepatic artery; CHD, common hepatic duct; HV, left hepatic vein; IHVC, infrahepatic vena cava; LPV, left portal vein; PV, portal vein; Remnant RL, remnant canine right liver lobe segment; RPV, right portal vein.
Fig 2. Serum laboratory values after reperfusion. CVF, cobra venom factor.
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Fig 3. Serum values after reperfusion. aPTT, activated partial thromboplastin time; FDP, fibrinogen degradation product; PT, prothrombin time.
Fig 4. (A) Healthy pig liver after harvest. (B) Xenograft 60 minutes after reperfusion in the control group. (C) Xenograft 60 minutes after reperfusion in the treatment group. (D) Remnant dog liver 60 minutes after reperfusion.
PIG TO CANINE HEPATIC XENOTRANSPLANTATION MODEL
DISCUSSION
The Pig-to-canine partial auxiliary liver xenotransplantation model is feasible. The donor pig body weight was about 5 kg less than the recipient. The pig suprahepatic vena cava was about 2.2 cm in greatest diameter, and the canine left hepatic vein was 2 cm, which was widened with oblique incision. The pig portal vein diameter was approximately 1.8 cm, and the canine left portal vein was about 1.3 cm, which was also widened with oblique incision. There were rapid intravascular biochemical changes which is reflecting hyperacute rejection. However, there was no severe aberrant wound bleeding or anastomosis site bleeding. There was no microscopic intravascular coagulation suggestive of hyperacute rejection. Although intravascular biochemical changes were less severe in the CVF⫹Gd treatment group, more cases are needed to obtain statistical significance. In the CVF⫹Gd treatment group, the pathologic findings (eg, patch hepatocyte necrosis) were less severe. Inasmuch as there were no corresponding vascular pathologic findings, these findings could
2759
not be a direct effect of CVF⫹Gd treatment. Other factors such as ischemia-reperfusion injury should be considered to explain these finding. REFERENCES 1. Byrne G, McCurry K, Martin M, et al: Transgenic pigs expressing human CD59 and decay accelerating factor produce an intrinsic barrier to complement-mediated damage. Transplantation 63:149, 1997 2. Cozzi E, Tucker AW, Langford GA, et al: Characterization of pigs transgenic for human decay- accelerating factor. Transplantation 64:1383, 1997 3. Lin SS, Hanaway MJ, Gonzalez SG, et al: The role of anti-Gal(alpha1–3)Gal antibodies in acute vascular rejection and accommodation of xenografts. Transplantation 70:1667, 2000 4. Loss M, Vangerow B, Schmidtko J, et al: Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model. Xenotransplantation 7:186, 2000 5. Nakajima R, Nakajima S, Nagata S, et al: Analysis of hyperacute rejection in newborn pig to dog lung xenotransplantation. Transplant Proc 32:1131, 2000