Demetris, AJ

Fung, JJ

Ildstad, ST

Qian, S

Starzl, TE

English

PubMed

D-Scholarship@Pitt

Orthotopic liver transplantation in the mouse

Name not available

Qian, SG

Demetris, AV

----~-- --~--- ------562 TRANSPLANTATION Vol 52,No. 3 Departments of Surgery and Pathology Washington University School of Medicine St. Louis, Missouri REFERENCES 1. Paul LC, van Es LA, de La Riviere GB. Eerniue G, DeGraeff J. Blood group B antigen on renal endothelium as the target for rejection in an ABO-incompatible recipient. Transplantation 1978;26:268. 2. Dejelo CL, William TC. B-cell cross-match in renal transplanta-tion. Lancet 1977; 2: 241. 3. Cerilli J, Brasile L. Endothelial cell alloantigens. Transplant Proc 1980; 12: 37. 4. Stastny P. Endothelial-monocyte antigens. Transplant Proc 1980; 12: 32. 5. Pierce JC, Kay S, Lee HM. Donor specific IgG antibody and the chronic rejection of human renal allografts. Surgery 1975; 78: 14. 6. Paul LC, Baldwin WM, van Es LA. Transplantation antigens on renal endothelium. Neth J Med 1982; 25: 208. 7. Mohanakumar T, Waldrep JC, Phibbs M, Mendez-Picon G, Ka-plan AM, Lee HM. Serological characterization of antibodies eluted from chronically rejected human renal allografts. Trans-plantation 1982; 32: 61. 8. Klassen J, Kano K, Milgrom F, et al. Tubular lesions produced by autoantibodies to tubular basement membrane in human renal allografts. Int Arch Allergy Appl Immunol 1973; 45: 675. 9. Jordan SC, Barkley SC, Lemire JM, Sakai RS, Cohen A, Fine RN. Spontaneous anti-tubular-basement-membrane antibody pro-duction by lymphocytes isolated from a rejected allograft. Trans-plantation 1986; 41: 173. 10. Joyce S, Flye MW, Mohanakumar T. Characterization of kidney cell-specific, non-major histocompatibility complex alloantigen using antibodies eluted from rejected human renal allografts. Transplantation 1988; 46: 362. 11. Johnson AH, Poo PA, McKeown PT, et al. Analysis of sera specific for human B cells. Transplant Proc 1977; 9: 147. 12. Neefjes JJ, Dolriadia I, Stam NJ, Becker CJ, Ploegh HL. An analysis of class I antigens of man and other species by one dimensional IEF and immunoblotting. Immunogenetics 1986; 23: 164. 13. Clen JCS. Glycoprotein detection in nitrocellulose transfers of electrophoretically separated protein mizturea using concana. valin A and perozidase: application to arenavirua and tlavivirua proteins. Anal Biochem 1982; 127: 389. 14. Joyce S. Structural characterization of human kidney specific non-MHC alloantigen [Ph.D. thesis]. Richmond, VA: Virginia Com. monwealth University, 1988. 15. Salinovich 0, Montelaro RC. Reversible staining and peptide map· ping of proteins transferred to nitrocellulose after separating by sodium dodecylsuifate polyacrylamide gel electrophoresis. Anal Biochem 1986; 156: 341. Received 22 June 1990. Accepted 30 January 1991. ORTHOTOPIC LIVER TRANSPLANTATION IN THE MOUSE Heart and kidney transplantation in the mouse has been well established (1,2), but orthotopic liver transplantation has not been reported. Although the mouse is one-tenth the size of the rat, there are many advantages to using the mouse for immu-nologic research. The mouse genome has been more thoroughly characterized than the rat or any other species of mammal. The mouse H-2 system also bears a striking resemblance to the human HLA system. In addition, there are numerous geneti-cally defined inbred strains and wealth of monoclonal antibod-ies that are commercially available for mouse investigations. A key development in rat orthotopic liver transplantation (3) was the introduction by Zimmerman et al. (4) and Kamada and Caine (5), of the cuff technique instead of suture for some of the venous vascular anastomoses. This method shortened the clamping time of the portal vein and increased survival. We have applied this principle to mouse orthotopic liver trans-plantation. In our pilot studies, more than 40 syngeneic mouse liver replacements were performed before 6 long-term survivors were obtained. The experience reported here is with the next 48 attempts, in which the surgical success rate was 83%. Male inbred syngeneic mice 10-12 weeks old (25-32 g), from Jackson Laboratory, Bar Harbor, ME, were used as size-matched donors and recipients under methoxyflurane (2.2-dichloro-1,1-difluoroethyl methyl ether) anesthesia. The strains used were B6AF1 (27 pairs), C57BL/6 (11 pairs). and BALB/c (10 pairs). Clean but not sterile operative technique was used, and all procedures were performed under the oper-ating microscope with 4-6.4x magnification. Donor operation. After shaving and disinfecting the abdomen with Betadine or alcohol, a complete midline incision was made. Four small retractors and a retracting clamp on the xiphoid process provided exposure. The bile duct was transected. The pyloric, splenic, right adrenal, and right renal veins, as well as the hepatic artery were ligated. Heparin (100 units) diluted to 0.3 ml with lactated Ringer's solution was slowly injected into the penile vein. After cross-clamping the thoracic aorta and dividing the suprahepatic vena cava close to the diaphragm, the liver was perfused with 1 ml cold lactated Ringer's solution, given through the abdominal aorta. Since the hepatic artery was already ligated, the liver perfusion was from the splanchnic venous return. The liver and surrounding tissues were kept cool with frequent rinses of cold lactated Ringer's solution. The infrahepatic vena cava was divided at the level of the left renal vein, leaving a sufficient length for cuff preparation. The freed liver was then placed into a container of cold lactated Ringer's solution for further preparation. The cystic duct was ligated and the gallbladder removed. The infrahepatic vena cava and the portal vein were cleaned for insertion through Teflon tubes (outer diameter 1.7 mm for vena cava, 1.2 mm for portal vein), using a cuff length of 1.5 mm and an equal vein length, which was folded back and tied (Fig. 1). These steps were facilitated by a stabilizing clamp. Recipient operation. Through a midline incision. the bile duct, hepatic artery, and right adenal vein were ligated and divided. The liver ligaments were incised and the suprahepatic vena cava was encircled with a ligature, which was used to pull the liver and diaphragm down for application ota Santinsky clamp on the suprahepatic vena cava. When the liver was removed, the infrahepatic vena cava, portal vein, and suprahepatic vena cava were cross-clamped, in that order. Maximum length was retained with the recipient to facilitate the venous reconnec-tions. The suprahepatic vena cava anastomosis was constructed first with a continous 10-0 nylon suture (Dermalon, Davis & Geck Inc.). A one-suture anastomosis technique was used to September 1991 BRIEF COMMUNICATIONS 563 FIGURE 1. Method of venous cuff preparation used for the portal vein and infrahepatic vena cava. Note that the liver is submerged in cold solution during these maneuvers. FIGURE 2. Continuous suture anastomosis of the suprahepatic vena cava, using a one-suture technique for manipulation and exposure of the posterior vessel walls. aid in manipulating and visualizing the posterior anastomotic wall (Fig. 2). Next, the graft portal vein cuff was inserted into a venotomy in the anterior wall of the recipient portal vein which was kept on tension with a clamp and inflated with injected saline. A silk tie was placed to secure the vein over the cuff and the liver was revascularized by removing the supra-hepatic and portal venous clamps. Portal venous occlusion was always for less than 20 min. The infrahepatic vena cava cuff was inserted in a ;·similar way, taking care to avoid excessive retrograde bleeding in the process. No attempt at rearteriali-zation was made. Biliary continuity was restored by tying the ducts over a 4-mm polyethylene tube stent (00 of 0.61 mm, Becton Dickinson; Parsippany, NJ). Blood lost during opera-tion was replaced with lactated Ringer's solution. The abdomen was closed in two layers. Single intramuscular injections of 5 mg cefamandol nafate and 60,000 units Bicillin were given and the mice were kept under a warming lamp until they became active. Food and water were allowed ad libitum. Eight mice were bled from the retroorbital sinus weekly for the first month and monthly thereafter postoperatively for determination of serum glutamic oxaloacetic transaminase"al-100 90 80 70 l 60 -' c 50 :> :;: a: 40 ::;) en 30 B6AF, '0 _ ~Bre 11 20 BALBlc 10 10 0 0 2 4 6 8 10 12 14 WEEKS FIGURE 3. Life survival (actual) after syngeneic liver replacement in three strains of mice. kaline phosphatase, bilirubin, total protein, and albumin. Ani-mals that died from complications of the operation were autop-sied, and histopathologic analyses were obtained when the cause of death was not readily apparent. In long-term survivors, tissues for histopathology were obtained by reoperation and wedge biopsy. Tissues were fixed in neutral buffered formalin, embedded in paraffin, sectioned at 4 p. and stained with he-matoxylin and eosin. Of the 48 recipients, 40 (83.3%) lived for more than one week, and 27 (67%) lived more than 100 days. The longest survivors have been followed for more than 12 months. The survival curves were similar in the 3 strains (Fig. 3). The deaths in the first postoperative week were from tech-nical causes. The causes of death of the 13 recipients that died between 8 and 100 days were bile duct complications (4 exam-ples), peritonitis (3), pneumonia (3), phlebotomy for blood sample collection (2), and unknown (1). The delayed deaths were concentrated in the second and third weeks (Fig. 3), and after this only a single mouse was lost (from a phlebotomy). Because 2 mice died from the phlebotomies, one early and one late, only 6 provided enough serial samples to be included. The SGOT levels were elevated after transplantation, as were the serum bilirubins, but these returned to or toward normal except in an animal that died of bile duct complications. In contrast to the other biochemical tests, the alkaline phospha-tases tended to become worse with time and were above the upper limits of normal in all animals tested for the full 100 days. Total protein and albumin levels were normal except in the animals that died of bile duct complications. Histopathologic studies showed that mice that died within 3 weeks had areas of scattered coagulation necrosis, most prom-inently in the hepatic hilum. Abscesses of variable sizes were in or around the infarcted areas. Mononuclear infiltrates were not seen in the viable liver but the liver sinusoids frequently contained neutrophils. The extrahepatic organs were unre-markable. Two mice had open liver biopsy at 49 and 56 days, and a third was killed at 133 days. Grossly, the livers appeared normal. Histopathologically, the livers had no major changes (Fig. 4), although there were subtle, usually focal, abnormali-564 TRANSPLANTATION VoL 52, No.3 FIGURE 4. Syngeneic liver graft of mouse killed 133 days postoper-atively (H&E; original magnification: X40). ties. Many of the triads had mild fibrosis and/or mild ductular proliferation with a light lymphoplasmocytic inflammatory in-filtrate. The duct epithelium frequently showed apoptosis and vacuolization. Hepatic arteries were difficult or impossible to identify in most of the triads. There was no evidence of choles-tasis. To our knowledge, this is the first description of orthotopic liver transplantation in the mouse. Although the surgical prin-ciples and techniques of OLT in the mouse are similar to those in the rat, the procedure is more difficult because of the smaller size. The most troublesome step was the suprahepatic vena cava anastomosis. This was facilitated by using one-suture anastomosis technique for the purpose of manipulation and ensurance of lumen patency. The anastomosis, which required 12-16 min, was performed with continuous 10-0 Dermalon because of the softness and flexibility of this suture. In our developmental series, and in a few of the animals in the definitive series, an anhepatic period greater than 20 min was incompatible with consistent success. This may reflect an in-tolerance of the mouse to hepatic ischemia, to venous conges-tion of the splanchnic and systemic systems, or to both. In the rat, an anhepatic period less than 26 min can be routinely tolerated (5). The lethal surgical complications were from defective revas-cularization of the portal vein. As with the rat (4, 5), failure to provide an arterial blood supply did not jeopardize long survival. However, degenerative changes in the biliary epithelium and other subtle histopathologic abnormalities in the portal triads may have been the consequence of this omission. It is notewor-thy that the canalicular enzyme, alkaline phosphatase, was the one biochemical index that did not become normal during the 100-day period of observation. Notwithstanding this potential imperfection, the mouse model should be useful in the future study of liver transplan-tation. Since numerous genetically defined inbred strains-including congenic strains of mice-are available, immunologic studies should be possible with greater precision than has been feasible until now. The resulting new information obtained may help to explain why the liver is privileged relative to other organs and why it may have a protective effect on other concomitantly transplanted organs (5). The availability of well-defined monoclonal antibody reagents will allow tracer studies and identification of cells involved in allorecognition and rejec-tion or the tolerance induction which is incongruously easy after liver transplantation (6). The prospect of evaluating monoclonal antibodies for therapeutic purposes is equally in-viting. Finally, several unique liver disease models, such as mouse viral hepatitis, that are analogs of human disorders, can be studied in greater detail (7, 8). SHIGUANG QIANI JOHN J. FUNG ANTHONY J. DEMETRIS2 SUZANNE T. ILDSTAD THOMAS E. STARZL Departments of Surgery and Pathology School of Medicine University of Pittsburgh Pittsburgh, Pennsylvania 15261 1 Address correspondence to: Shiguang Qian, M.D., Department of Surgery, Transplantation Laboratory, E1540A Biomedical Science Tower, University of Pittsburgh, Pittsburgh, PA 15261. 2 Department of Pathology. REFERENCES 1. Corry KJ, Winn HJ, Russell PS. Heart transplantation in congenic strains of mice. Transplant Proc 1973; 5: 733. 2. Skoskiewicz M, Chase HJ, Russell PS. Kidney transplants between mice of graded immunogenetic diversity. Transplant Proc 1973; 5: 721. 3. Lee S, Charter C, Chandler JG, Orloff MJ. A technique for ortho-topic liver transplantation in the rat. Transplantation 1973; 16: 664. 4. Zimmerman FA, Butcher GW, Davies HS, Brons G, Kamada N, Turello O. Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic response to fully allo-geneic liver grafts. Transplant Proc 1979; 11: 571. 5. Kamada N, Caine RY. Orthotopic liver transplantation in the rat: technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 1979; 28: 47. 6. Murase N, Kim DG, Todo S, Cramer DV, Fung JJ, Starzl TE. Suppression of allograft rejection with FK506: I. Prolonged car-diac and liver survival in rats following short course therapy. Transplantation 1990; 50: 739. 7. Dindzans VJ, Skamene E, Levy GA. Susceptibility/resistance to mouse hepatitis virus strain 3 and macrophage procoagulant activity are genetically linked and controlled by two non-H-2 linked genes. J Immunol 1986; 137: 2355. 8. Nesbitt MN, Skamen E. Recombinant inbred mouse strains derived from A/J and C57 bl/6J a tool for the study of genetic mecha-nisms in host resistance to injection and malignancy. J Leukocyte BioI 1984; 36: 357. Received 14 November 1990. Accepted 23 January 1991. & 

Orthotopic liver transplantation in the mouse.

http://d-scholarship.pitt.edu/4628/1/31735062117936.pdf

Orthotopic liver transplantation in the mouse

Abstract

Similar works

Full text

Available Versions

D-Scholarship@Pitt

Name not available

Name not available

D-Scholarship@Pitt