Limb transplantation in rats: Immunosuppression with FK-506

Limb Transplantation in Rats: Immunosuppression With FK-506 Zhao Min, MD, Neil F. Jones, MD, Los Angeles, CA The efficacy of a new immunosuppressive agent, FK-506, to prevent the rejection of limb transplants was investigated across a major histocompatibility barrier in rats and compared with Cyclosporine. We performed 129 limb transplants between donor ACI rats and recipient Lewis rats. The mean rejection time of the skin component of limb allografts without immunosuppression was 7 days. Animals receiving a 14-day course of Cyclosporine 25 mg/kg had a mean rejection time of 30 days. In contrast, animals receiving a 14-day course of FK506 1 mg/kg had a mean rejection time of 54 days and those receiving a 14-day course of FK-506 2 mg/kg had a mean rejection time of 122 days. Intermittent administration of FK-506 twice weekly further prolonged the mean rejection times to 149 days in animals receiving 1 mg/kg and to 296 days in animals receiving 2 mg/kg. (J Hand Surg 1995;20A:77-87.)

Experimental limb transplantation has been attempted using various immunosuppressive regimes including parabiosis, ~ blood transfusion, 2"3 antilymp h o c y t e serum, 4 6 - m e r c a p t o p u r i n e , 5"6 azathiop r i n e , 3,5 s t e r o i d s , 3'6"7 and Cyclosporine (CyA). 7-22 C y c l o s p o r i n e has had an e n o r m o u s impact on human vital organ transplantation. In experimental limb transplantation during the past decade, it has been shown to prolong the survival of rat limb allografts, depending on the dosage regimen and the histocompatibility of the donor and recipient rats. 7-~9 However, for extended survival of a composite limb transplant, continuous administration of cyclosporine (CyA) seems to be required. ~~ Unfortunately, administration of CyA is associated with

From the Division of Plastic and Reconstructive Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA. Received for publication Aug. 19, 1993; accepted in revised form July 22, 1994. Although the author of authors have not received or will not receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article, benefits have been or will be received but are directed solely to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors are associated. Reprint requests: Neil Ford Jones, MD, UCLA Hand Center, Division of Plastic and Reconstructive Surgery and Department of Orthopaedic Surgery, 200 UCLA Medical Plaza, Los Angeles, CA 90024-6960.

well-recognized side effects, such as nephrotoxicity and hepatotoxicity. Several new drugs including FK-506, RS-61443, and Rapamysin as well as combination immunosuppressive therapy are currently undergoing experimental and clinical evaluation. FK-506, a potent new immunosuppressive agent, was isolated from Streptomyces tsukubaensis in Osaka, Japan in 1984. 23 FK-506 is a macrolide antibiotic with a different chemical structure but a similar mechanism of action to Cyclosporine. FK-506 is reported to be about 10-100 times more potent than Cyclosporine, but has a lower incidence of side effects. This study investigates the effectiveness of FK506 in preventing rejection of limb transplantation in rats across an extremely strong histocompatibility barrier and to compare its efficacy with Cyclosporine.

Materials and Methods Inbred strain Lewis (RTI-1) rats and ACI (RT1a) rats (Harlan Sprague D a w l e y , Indianapolis) weighing 225-250 gms (mean 233 gms) and 8-12 weeks of age were used as recipients and donors, respectively, for orthotopic limb transplantation. This combination represents a very strong histocompatibility mismatch. One group of animals, h o w ever, consisted of male animals 6-7 months old with a mean body weight of 346 gms. Under general anThe Journal of Hand Surgery

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Min and Jones / Limb Transplantation in Rats: Immunosuppression With FK-506

aesthesia using intramuscular Ketamine 50 mg/kg body weight, the donor hind limb of an ACI rat was transected at mid femur. All the skin of the donor limb was removed except the skin over the foot. In the recipient Lewis rat, the hind limb was again amputated at mid-femur level, but the skin envelope was preserved down to the ankle (Fig. 1A & B). The donor foot skin acted as a monitor of the circulation and eventual rejection of the allograft. The skin envelope of the recipient limb provided coverage of the donor knee joint and musculoskeletal tissues after rejection of the foot skin had occurred. 24,25 Osteosynthesis was achieved orthotopically using an 18 gauge needle as an intramedullary rod. The femoral artery, vein, femoral nerve, and sciatic nerve were repaired using standard microsurgical techniques with 10-0 nylon sutures. The muscles of the thigh were approximated and the skin sutured (Fig. 1C). The mean ischemic time for the donor limb was 60 minutes. A single dose of Gentamicin 2000 units was given intramuscularly immediately after surgery and 15 cc of normal saline was infused intraperitoneally to compensate for fluid losses during surgery. A plastic neck collar was used for 2-3 weeks to prevent self-mutilation of the anesthetic foot. In addition to control autografts in 15 Lewis rats (Group 0), 114 limb transplants were performed, which were assigned to 10 groups based on the type and dosage of immunosuppression (Table 1). FK506 was dissolved in saline and administered intramuscularly. CyA suspended in an oil carrier was administered subcutaneously. After surgery, clinical, histologic, and radiologic evaluations of the transplanted limbs were performed. The animals were weighed weekly and the limb allografts were observed daily for signs of rejection of the donor foot skin. The foot skin of the transplanted limb was always compared with the normal contralateral limb. Skin rejection, a progressive process, occurred in a reproducible sequence of erythema, edema, epidermolysis, desquamation, exudation, eschar formation, and mummification (Figs. 2 & 3). The onset of skin rejection was rigidly defined when the first sign beyond erythema and edema occurred. The animals ambulated satisfactorily on their transplanted limb and gradually developed weight-bearing gait. X-ray films were obtained approximately 8 weeks after surgery and before the rats were killed. Biopsies of the skin, muscle, bone osteosynthesis, and knee joint were obtained for histologic examination and stained with hematoxylineosin and trichrome stains. The histologic changes of the skin, muscle, bone, and knee joint from the transplanted limb were graded by an independent

Figure I. (A) Donor ACI rat (left) and recipient Lewis rat (right). (B) Harvesting of two donor ACI hind limbs, leaving only the skin over the ankle and foot. (C) Appearance immediately after transplant with coverage of the musculoskeletal tissues of the donor ACI limb within the thigh skin envelope of the recipient Lewis rat. pathologist as 0, normal; 1, mild rejection; 2, moderate rejection; and 3, severe rejection. Survival times for the experimental groups were analyzed statistically using one-way analysis of variance and generalized Wilcoxon test. Beta technique was used to stabilize variance for data transforma-

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Table 1. Experimental Groups

Group 0 1 2 3 4 5 6 7 8 9

Number of Animals 15 6 10 11 9 10 10 9 8 9

Sex F F F M (young) M (old) F F F F F

10

10

F

Immunosuppression Autografts No immunosuppression FK-506 FK-506 FK-506 FK-506 FK-506 FK-506 CyA FK-506 FK-506

tion. P values less than .05 were considered significant.

Results Of 114 successful limb transplants, 22 developed postoperative complications: early death within 4 days (9); late death within 30 days (7); self-mutilation (4); and technical failure (2). This left 92 animals with successful limb transplants for analysis. All 15 autografts survived uneventfully and were killed at 3 months for histologic evaluation. Following surgery their foot skin became pink and developed slight edema, which resolved within 3-4 days~ In contrast, the skin of the donor limb allograft was the first target of the recipient immune response. Once the skin was rejected, the whole transplant underwent progressive rejection culminating in necrosis and mum-

Dosage --1 mg/kg/d 1 mg/kg/d 1 mg/kg/d 1 mg/kg/d 2 mg/kg/d 20 mg/kg single dose 25 mg/kg/d 1 mg/kg/d 1 mg/kg twice weekly 2 mg/kg/d 2 mg/kg twice a week

Duration (Day) --0-13 0-13 1-14 3-16 0-13 3 0-13 3-16 1-14

mification. In this study, the time at which the specific signs of rejection of the donor foot skin occurred was used as the parameter of rejection of the entire transplant. The skin of the limb allografts in group 1 receiving no immunosuppression rapidly developed signs of gross rejection with progressive edema and exudation in 6-8 days (mean rejection time [MRT], 7 days). This was rapidly followed by eschar formation, total necrosis, and mummification within 2-3 weeks (Fig. 2). In contrast, FK-506 significantly prolonged the survival of all the immunosuppressed limb allografts in groups 2, 3, 4, 5, 6, 7, 9, and 10, (range, 30-599 days) (Table 2 and Figs. 4 & 5). Before rejection, the limb allografts appeared healthy with normal hair and nail growth. MRT did not differ significantly among groups 2, 3, 5, and between groups 3 and 4. These data indicate that delaying

Figure 2. Rejection of limb allografts without immunosuppression showing erythema and edema 5 days after transplant (A) and mummification at 20 days after transplant (B).

80 Min and Jones / Limb Transplantation in Rats: Immunosuppression With FK-506

Figure 3. Postoperative stages of rejection of individual limb allografts immunosuppressed with FK-506: (A) edema at 101 days; (B) edema and erythema at 217 days; (C) desquamation at 67 days; (D) crust and exudation at 43 days; (E) removal of the crust revealing viable deeper tissues; (F) total mummification at 71 days.

the administration of FK506 until day 1 or day 3 following the transplant was as effective as beginning immunosuppression immediately after transplantation on day 0. The sex and age of the recipient rat had no significant influence on its immune response. The fact that the old male rats (group 4: MRT, 58 days) accepted the allografts for considerably longer time than the young female rats (group

2: MRT, 40.30 days; p < .05) could be explained by the combined effect of sex and age on the outcome of the allografts. FK-506 demonstrated a dose-dependent response, with a short-term course of FK506 2 mg/kg prolonging the mean rejection time in group 6 animals to 122 days, a significant difference from any other group receiving short-term FK-506 1 mg/kg ( g r o u p s 2 - 5 : M R T , 4 0 - 4 5 d a y s ; p <

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tense than other treated groups. Once rejection occurred, the donor foot became severely swollen with more exudation and breakdown of the entire skin and subcutaneous tissues. A 14-day course of CyA 25 mg/kg/d prolonged the MRT in group 8 animals to 30 days, which was, however, much shorter than all groups receiving FK-506 (groups 2-6), indicating that in this donor-recipient strain transplant, FK-506 is more effective in prolonging limb allograft survival than CyA, (group 8 vs. groups 2-5, p < .05). Long-term intermittent immunosuppression with FK-506 was superior to the same dose used for a short-term 14-day course (Figs. 3A, 6B). Group 9 animals receiving long-term intermittent FK-506 l mg/kg had an MRT of 149 days c o m p a r e d with groups 2-5 receiving only 14 days of FK-506 1 mg/ kg with an MRT of 40-45 days (p < .001). Similarly group 10 animals receiving long-term intermittent FK-506 2 mg/kg had an MRT of 296 days compared with group 6 animals receiving only 14 days of FK-506 2 mg/kg who had an MRT of 120 days (p < .001). All the animals in group I0 died of bacterial pneumonia rather consistently between 273-334 days, except for one animal who died accidentally 107 days after transplant without any signs of rejection. There were no signs of infection or drug toxicity in any of the transplanted animals. Primary wound healing occurred in all animals and the femoral vessels remained patent during the observation period. Weight loss occurred immediately following transplantation, but the animals progressively regained their baseline weight. Any subsequent weight loss was then associated with skin rejection in all animals

Table 2. Mean Rejection Times (MRT) of Limb Allografts Group

l 2 3 4 5 6 7 8 9 10

Day of Rejection

MRT • SD

6, 7, 6, 7, 8, 7 37, 33, 30, 40, 41, 47, 42, 35, 57, 41 57, 63, 39, 52, 45, 35, 45, 36, 43, 48, 32 42, 46, 82, 112, 51, 57, 56, 46, 33 49, 32, 46, 57, 41, 60, 56, 36, 30, 47 84", 76, 87, 64, 599, 67, 56, 58, 55, 74 44, 83, 39, 144, 63, 65, 47, 38, 56 31, 32, 30, 30, 28, 34, 27, 31 50, 74, 222, 223, 116, 287, 109, 192, 70 318", 293", 315", 273", 303", 239", 334*, 317", 273*.

7 • 0.75 40 • 7.65 45 • 9.57 58 • 24.27 45 • 10.54 122 • 159.35 64 • 33.19 30 • 2.20 149 • 83.71 296 • 29.78

* Number of days after transplant when the animal died or was sacrificed without signs of rejection. .05-.001). Moreover, one animal in this group survived completely free of rejection of its transplant for 599 days (Fig. 6A). The mean rejection time of animals receiving a large dose of FK-506 20 mg/kg (group 7) was 64 days, comparable to all the short-term courses of FK-506 1 mg/kg (groups 2-5: MRT, 40-58 days). The animals in this group tolerated the high dose of FK-506 without any mortality and looked healthier than other animals receiving daily injection of the drug. However, the rejection process of the allografts in group 7 seemed to be more rapid and inMean Rejection Time (days) 350 r 300 250 200 150 100

50[ 0

1

2

3

4

81

5

6

7

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9

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Figure 4. Mean rejection times of limb allografts in the 10 groups of animals.

82

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Figure 5. (A) Percentage survival of limb allografts in group 2 (FK-506 lmg/kg for 14 days), group 6 (FK-506 2mg/kg for 14 days), and group 10 (FK-506 2mg/kg for 14 days followed by 2mg/kg twice weekly). (B) Percentage survival of limb allografts in group 2 (FK-506 lmg/kg for 14 days), group 9 (FK-506 lmg/kg for 14 days followed by lmg/kg twice weekly), and group 10 (FK-506 2mg/kg for 14 days followed by 2mg/kg twice weekly).

(Fig. 7). X-ray films revealed union of the donor femur to the recipient femur at the site of osteosynthesis approximately 8 weeks after surgery. Xray films taken during long-term follow-up examination confirmed the preservation of the knee joint (Figs. 8). Relatively normal growth of the transplanted limb was observed. Histologic evaluation revealed bony healing and preservation of the articular cartilage within the knee joint of long-term survivors (Fig. 9).

Discussion Before the introduction of specific immunosuppressive agents, experimental limb allografts were attempted by inducing immunologic tolerance in rats by parabiosis or by infusing recipient-derived antidonor antiserum 1"4 and in dogs by subtotal blood exchange in the newborn. 2 Limb transplantation was then attempted in dogs using 6-mercaptopurine and azathioprine 5 or by

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Figure 6. (A) Long-term survival 243 days after transplant after a 14-day course of FK-506 2mg/kg. (B) Long-term survival 168 days after transplant with intermittent immunosuppression with FK-506 1 mg/kg twice weekly. longed survival of limb allografts from Lewis-Brown Norway donor rats to Lewis recipient rats. s Dose response testing showed that a 20-day course of CyA at either 25 mg/kg or 8 mg/kg prolonged survival to 101 days compared with 18 days in control non-immunosuppressed recipients. 9 Indefinite survival could be achieved in some animals across this same Lewis-Brown Norway---~Lewis mismatch after an initial 20-day course of CyA 8 mg/kg followed by either 8 mg/kg subcutaneously twice weekly or 8 mg/ kg/day orally. ~~ Some of these long-term survivors developed lymphoid chimerism in that the lymphocytes from the peripheral blood and spleen of the L e w i s recipients c o n t a i n e d 20% L e w i s - B r o w n

using azathioprine and hydrocortisone in combination with splenectomy and thymectomy followed by further induction of immune tolerance using donor splenic cells or exchange transfusions. 3 Doi compared various early immunosuppressive agents such as azathioprine, 5-mercaptopurine, and prednisone either alone or in combination in rat limb transplants across the Wistar--->Fisher 344 barrier and demonstrated that azathioprine and prednisone were the only combination effective in prolonging rejection. 6 After the introduction of CyA into clinical vital organ transplantation in 1976,26 this drug was investigated in experimental limb transplantation. A 20day course of CyA 25 mg/kg subcutaneously pro-

weight(gms)

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Figure 7. Postoperative weight changes of animals 15 and 96. Initially, all animals lost weight following transplantation but subsequently regained their baseline weight. Animal 15 shows a secondary weight loss beginning on the 42nd day corresponding to clinical signs of rejection of the donor foot skin. In contrast, animal 96 continued to maintain its weight and did not show any signs of rejection 149 days after transplant.

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Min and Jones / Limb Transplantation in Rats: Immunosuppression With FK-506

A Figure 8. (A) X-ray film showing healing of the donor femur to the recipient femur 60 days after transplant. (B) X-ray film showing good bony healing of a long-term limb allograft 261 days after transplant.

N o r w a y d o n o r - d e r i v e d cells, ll S e v e r a l o t h e r a u t h o r s have i n v e s t i g a t e d the e f f e c t i v e n e s s of CyA in preventing the rejection of limb allografts using d i f f e r e n t s t r a i n s and d i f f e r e n t dosage regimes: BUF--~Lewis, 12 ACI---~Lewis, x3 Fisher--~ Lewis. 7,14,15 In a series of 90 limb transplants, Hotokebuchi et al. were able to demonstrate the survival of limb allografts across a minor mismatch barrier Lewis---~F344 for more than 1 year using either a short-term 16-day course or a longterm twice w e e k l y c o u r s e of C y A 25 mg/kg. 16

Figure 9. Specimen showing bony healing of the donor femur to the recipient femur after the animal was killed 188 days after transplant.

Across a major mismatch Brown Norway-+F344, however the donor foot skin only survived for 45 days with short-term immunosuppression and 56 days with long-term immunosuppression compared with rejection at 11 days in the control nonimmunosuppressed rats. Cyclosporine has also been investigated in experimental limb transplantation in baboons and monkeys. 2~ Two groups have investigated the use of FK-506 in experimental limb transplantation. Kuroki et al.27"28 have demonstrated improved survival of limb allografts across the Lewis-+PVG barrier in those animals receiving FK-506 0.64 mg/kg for 14 days compared with those rats receiving CyA 15 mg/kg for a similar period. Arai et al. 29"30 in a series of 69 limb transplants from Brown Norway---~F344 rats were able to demonstrate prolonged survival to 150 days using a 14-day course of FK-506 1 mg/kg. Furthermore, a single dose of FK-506 10 mg/kg or 50 mg/kg on the day of transplantation also prolonged survival to 51 and 104 days respectively. Even delayed treatment with a single dose of FK-506 10 mg/ kg on day 7 or day 10 after the transplant reversed the early signs of rejection. Long-term intermittent t r e a t m e n t w i t h FK-5063 mg/kg o n c e a w e e k achieved indefinite limb allograft survival although many of the rats developed Pneumocystis carinii pneumonia, suggesting the development of graftversus-host disease.

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The only other reports of immunosuppression in experimental limb transplantation have involved 15deoxyspergualin and RS-61443. A 10-day or 20-day course of 15-deoxyspergualin produced a modest prolongation of survival of limb allografts of 18 to 24 days across the Dark Agouti---~Lewis barrier. 3~ Benhaim et al. have investigated the effectiveness of RS-61443, the morpholinoethyl ester of mycophenolic acid, in rat limb transplantation. 3z Five of six animals treated continuously with RS-61443 30 mg/ kg/day showed no clinical or histologic evidence of rejection of the skin component of limb allografts when the animals were killed between 231 and 251 days after transplant. This was significantly more effective than animals receiving CyA 10 mg/kg for 20 days followed by twice a week. If the results of experimental limb transplantation in small animals are to be extrapolated to larger animals and eventually to potential clinical transplantation in humans, there is a pressing need for standardization of the donor-recipient histocompatibility mismatch and both clinical and histologic criteria of rejection, so that valid comparisons may be made between different immunosuppressive agents and different dosage regimes. Previously reported studies suggesting long-term survival of composite limb allografts have been performed across relatively minor histocompatibility barriers. In this study, ACI and Lewis strain rats were selected since they are acknowledged as the strongest immunologic mismatch short of performing a xenograft. Therefore, if an immunosuppressive agent will significantly prolong the survival of a limb allograft across the ACI--+Lewis barrier, this is much more valid experimentally and potentially clinically relevant than using a minor mismatch. The use of the ACI---~Lewis combination was discontinued in the study reported by Press et al. because CyA was unable to prolong the survival of a limb allograft in this model. 14 Various criteria for monitoring the rejection of the skin component of a limb allograft have been proposed, including erythema of the fool skin, a 5~ temperature drop in the limb, 8-1~ changes in the color of the limb, ~2 the presence of scaling, erythema or hair lOSS, 6"14'15 ability to wipe away the soft surface of the skin with the gentlest of touch or hardening or escharification of the entire surface, 16 complete skin necrosis,27 involvement of 80% of the skin with progressive dermal lesions, 5 or complete change to brown and necrotic skin. 7 With experience, observation of the rejection process reveals a sequential series of changes from e r y t h e m a to edema, blistering, desquamation, epidermolysis, and mummification and necrosis. We have rigidly

85

defined the onset of rejection as the first change beyond erythema and edema, although this may be more premature than the signs used in other studies. Erythema and edema are probably signs of incipient rejection and are potentially reversible. In two animals (not included in this study), the pre-rejection signs of erythema and edema were reversed by a single injection of FK-506 20 mg/kg, and frank rejection was postponed for a further 1-3 months. Using our rigid criteria of rejection, this study has shown that a 14-day course of FK-506 will prolong the survival of a limb allograft across the ACI---~ Lewis major histocompatibility barrier. Allograft survival is dose dependent, MRTs significantly prolonged in animals receiving FK-506 2 mg/kg when compared with animals receiving FK-506 1 mg/kg. The effectiveness of FK-506 was independent of the timing of the initial dose of the drug during the first 3 days after transplant. Comparison of those animals receiving a 14-day course of FK-506 (groups 2-5) with those animals receiving a 14 day course of CyA (group 8) demonstrated that FK-506 is significantly more effective in prolonging rejection than CyA. This study has also shown that a single dose of FK506 20 mg/kg given on day 3 is as effective in preventing the rejection of the skin component of a limb allograft as a 14-day course of FK-506 1 mg/kg, a finding similar to that reported by Arai et al. 29 When rejection did occur, however, the process was much more rapid and histology revealed chronic ongoing rejection in the musculoskeletal tissues. Administration of FK-506 twice weekly, especially at the higher dose of 2 mg/kg, has been very effective in allowing long-term survival of limb allografts. However, all the animals receiving FK-506 1 mg/kg twice weekly (group 9) eventually developed rejection, and all the animals receiving FK-506 2mg/kg twice a week lost weight and died without signs of skin rejection after approximately 300 days. This phenomenon is similar to that reported by Arai et al. 29 in that 6 of 8 of their long-term survivors died around 270 days after transplant from Pneumocystis carinii pneumonia. Histologic and bacterial examination of animals in group 10 of our series did not reveal Pneumocystis but did reveal bacterial pneumonia, suggesting graftversus-host disease. To our knowledge this study represents one of the largest series of limb transplants reported to date. However, these results cannot be extrapolated to justify clinical limb transplantation. Further work needs to be performed in larger animals. FK-506 is a very powerful immunosuppressive agent, which may perhaps play a role in the future in translating the transplantation of non-vital tissues into clinical reality as part of a "cocktail" of immunosuppressive agents.

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This work was supported by grants from the Plastic Surgery Educational Foundation and the AO/ASIF Research Commission. Cyclosporine was provided by the Sandoz Pharmaceuticals Corporation, and FK-506 was provided by the Fujisawa Pharmaceutical Company Ltd.

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30. Arai K, Hotokebuchi T, Miyahara H, et al. Prolonged limb allograft survival with short-term treatment with FK-506 in rats. Transplantation Proc 1989;21:31913. 31. Walter P, Menger MD, Theis J, Wolf B, Dickneite G. Prolongation of graft survival in allogeneic limb

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transplantation by 15-deoxyspergualin. Transplantation Proc 1989;21:3186. 32. Benhaim P, Anthony JP, Lin LYT, McCalmont TH, Mathes SJ. A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443. Transplantation 1993;56:911-7.